Methods to identify modulators of FKHL7 DNA-binding activity

ABSTRACT

Methods and compositions for treating a congenital heart disease and methods and compositions for prognosing or diagnosing a congenital heart disease in a subject are disclosed.

This application is a divisional application based on U.S. Ser. No.09/083,351, filed on May 22, 1998 and now U.S. Pat. No. 6,087,107, whichitself claims priority to provisional patent application 60/081,870,filed Apr. 15, 1998 and now abandoned.

1. BACKGROUND OF THE INVENTION

“Congenital heart disease” refers to defects in the heart and majorgreat vessels produced by abnormalities at various stages of fetaldevelopment and present at birth, but which may not be diagnosed untillater. The incidence of such anomalies is 1/120 live births (The MerckManual of Diagnosis and Therapy, 16^(th) Ed. (1992) p. 2051). “Atrialseptal defect” is form of congenital heart disease in which there is anopening in the septum that normally separates the atria. The typicalmurmur of atrial septal defect is usually present after age 1 yr., whenpulmonary blood flow has increased significantly.

Many congenital heart diseases have a genetic basis. However, surgeryoffers the only therapeutic option for many of these disorders. Inaddition, current identification and diagnosis of congenital heartdisease depends on the recognition of affected cardiac function, such asheart murmurs representing turbulent flow, altered systemic andpulmonary blood flow, shunting in either direction, and evidences ofaltered work load of the cardiac chambers. Routine history, physicalexamination, ECG, and chest x-ray are usually performed for specificanatomic diagnosis, with supportive and confirmatory data fromechocardiography, cardiac catheterization, angiocardiography and otherlaboratory data.

Improved therapies and diagnostics for genetically based congenitalheart diseases are needed.

2. SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery of anovel human gene, which encodes a novel human protein. These newlyidentified genes and proteins are referred to therein as “FKHL7”. FKHL7is a monomeric DNA binding protein that shares core binding site(RTAAYA) with four other FKHL7-like proteins. In addition, the forkheaddomain of this protein shows strong homology to the human gene, FKHL14and the mouse genes Fkh1 and Fkh14 by BLASTN analysis.

A 9.8 kb subclone of BAC471g19 was partially sequenced and determined tocontain the entire coding region of FKHL7 as well as 5′ and 3′untranslated sequences (SEQ ID NO. 1). The human FKHL7 coding sequenceis 1.7 kb in size and contains no introns. The 1659 bp open readingframe (SEQ ID NO. 3) encodes a 553 amino acid polypeptide (SEQ ID NO.2). The COOH-terminal domain contains several stretches of homopolymericruns of alanine and glycine. The FKHL7 coding region contains 5recognition sites for the restriction enzyme NotI. A BLASTN screen ofthe public dbEST database with the FKHL7 genomic sequence yields onlypartial human and mouse cDNA coverage of this gene. Based on theanalysis of cDNA clones identified in the public databases, there isevidence for the utilization of at least two different polyadenylationsignals within the 3′ untranslated region.

Human FKHL7 is most abundantly expressed during embryogenesis and of theadult tissue tested, significant expression was observed in adult eye,heart, kidney and lung, while relatively little to no expression wasobserved in adult skeletal muscle, spleen or liver.

In one aspect, the invention features isolated FKHL7 nucleic acidmolecules. In one embodiment, the FKHL7 nucleic acid is from avertebrate. In a preferred embodiment, the FKHL7 nucleic acid is from amammal, e.g. a human. In an even more preferred embodiment, the nucleicacid has the nucleic acid sequence set forth in SEQ ID NO. 1 or 3 or aportion thereof. The disclosed molecules can be non-coding, (e.g. aprobe, antisense, or ribozyme molecule) or can encode a functional FKHL7polypeptide (e.g. a polypeptide which functions as either an agonist orantagonist of at least one bioactivity of the human FKHL7 polypeptide).In one embodiment, the nucleic acid of the present invention canhybridize to a vertebrate FKHL7 gene or to the complement of avertebrate FKHL7 gene. In a further embodiment, the claimed nucleic acidcan hybridize with a nucleic acid sequence shown in FIG. 1 (SEQ ID NOS.1 and 3) or a complement thereof. In a preferred embodiment, thehybridization is conducted under mildly stringent or stringentconditions.

In further embodiments, the nucleic acid molecule is an FKHL7 nucleicacid that is at least about 70%, preferably about 80%, more preferablyabout 85%, and even more preferably at least about 90% or 95% homologousto the nucleic acid shouts as SEQ ID NOS. 1 or 3 or to the complement ofthe nucleic acid shown as SEQ ID NOS 1 or 3.

The invention also provides probes and primers comprising substantiallypurified oligonucleotides, which correspond to a region of nucleotidesequence which hybridizes to at least about 6, at least about 10, atleast about 15, at least about 20, or preferably at least about 25consecutive nucleotides of the sequence set forth as SEQ ID NO. 1 or SEQID NO. 3 or complements of the sequence set forth as SEQ ID NOS. 1 or 3or naturally occurring mutants or allelic variants thereof. In preferredembodiments, the probe/primer further includes a label group attachedthereto, which is capable of being detected.

For expression, the subject nucleic acids can be operably linked to atranscriptional regulatory sequence, e.g., at least one of atranscriptional promoter (e.g., for constitutive expression or inducibleexpression) or transcriptional enhancer sequence. Such regulatorysequences in conjunction with an FKHL7 nucleic acid molecule can providea useful vector for gene expression. This invention also describes hostcells transfected with said expression vector whether prokaryotic oreukaryotic and in vitro (e.g. cell culture) and in vivo (e.g.transgenic) methods for producing FKHL7 proteins by employing saidexpression vectors.

In another aspect, the invention features isolated FKHL7 polypeptides,preferably substantially pure preparations, e.g. of plasma purified orrecombinantly produced polypeptides. The FKHL7 polypeptide can comprisea full length protein or can comprise smaller fragments corresponding toone or more particular motifs/domains, or fragments comprising at leastabout 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 510, 520, 530 or 540 amino acidsin length. In particularly preferred embodiments, the subjectpolypeptide is capable of binding to an upstream region of a gene and/orotherwise regulating expression of a gene.

In a preferred embodiment, the polypeptide is encoded by a nucleic acid,which hybridizes with the nucleic acid sequence represented in SEQ IDNOS. 1 or 3. In a further preferred embodiment, the FKHL7 polypeptide iscomprised of the amino acid sequence set forth in SEQ ID NO. 2. Thesubject FKHL7 protein also includes within its scope modified proteins,e.g proteins which are resistant to post-translational modification, forexample, due to mutations which alter modification sites (such astyrosine, threonine, serine or asparagine residues), or which preventglycosylation of the protein, or which prevent interaction of theprotein with intracellular proteins involved in signal transduction.

The FKHL7 polypeptides of the present invention can be glycosylated, orconversely, by choice of the expression system or by modification of theprotein sequence to preclude glycosylation, reduced carbohydrate analogscan also be provided. Glycosylated forms can be obtained, for example,based on derivatization with glycosaminoglycan chains.

In yet another preferred embodiment, the invention features a purifiedor recombinant polypeptide, which has the ability to modulate, e.g.,mimic or antagonize, an activity of a wild-type FKHL7 protein.Preferably, the polypeptide comprises an amino acid sequence identicalor homologous to a sequence designated in SEQ ID NO. 2.

Another aspect of the invention features chimeric molecules (e.g.,fusion proteins) comprising an FKHL7 protein. For instance, the FKHL7protein can be provided as a recombinant fusion protein which includes asecond polypeptide portion, e.g., a second polypeptide having an aminoacid sequence unrelated (heterologous) to the FKHL7 polypeptide. Apreferred FKHL7 fusion protein is an immunoglobulin-FKHL7 fusionprotein, in which an immunoglobulin constant region is fused to an FKHL7polypeptide.

Yet another aspect of the present invention concerns an immunogencomprising an FKHL7 polypeptide in an immunogenic preparation, theimmunogen being capable of eliciting an immune response specific for anFKHL7 polypeptide; e.g. a humoral response, an antibody response and/orcellular response. In a preferred embodiment, the immunogen comprises anantigenic determinant, e.g. a unique determinant of a protein encoded bythe nucleic acid set forth in SEQ ID NO. 1 or 3; or as set forth in SEQID NO. 2.

A still further aspect of the present invention features antibodies andother binding proteins or peptides that are specifically reactive withan epitope of an FKHL7 protein.

The invention also features transgenic non-human animals which include(and preferably express) a heterologous form of an FKHL7 gene describedherein, or which misexpress an endogenous FKHL7 gene (e.g., an animal inwhich expression of one or more of the subject FKHL7 proteins isdisrupted). Such transgenic animals can serve as animal models forstudying cellular and/or tissue disorders comprising mutated ormis-expressed FKHL7 alleles or for use in drug screening. Alternatively,such transgenic animals can be useful for expressing recombinant FKHL7polypeptides.

The invention further features assays and kits for determining whetheran individual's FKHL7 genes and/or proteins are defective or deficient(e g in activity and/or level), and/or for determining the identity ofFKHL7 alleles. In one embodiment, the method comprises the step ofdetermining the level of FKHL7 protein, the level of FKHL7 mRNA and/orthe transcription rate of an FKHL7 gene. In another preferredembodiment, the method comprises detecting, in a tissue of the subject,the presence or absence of a genetic alteration, which is characterizedby at least one of the following: a deletion of one or more nucleotidesfrom a gene; an addition of one or more nucleotides to the gene; asubstitution of one or more nucleotides of the gene; a gross chromosomalrearrangement of the gene; an alteration in the level of a messenger RNAtranscript of the gene; the presence of a non-wild type splicing patternof a messenger RNA transcript of the gene; and/or a non-wild type levelof the FKHL7 protein.

FKHL7 mutations that are particularly likely to cause or contribute tothe development of congenital heart disease include mutations thatresult in an FKHL7 protein that lacks or contains a substantiallyimpaired FKHL7 gene.

FKHL7 mutations can be detected by: i) providing a probe/primercomprised of an oligonucleotide which hybridizes to a sense or antisensesequence of an FKHL7 gene or naturally occurring mutants thereof, or 5′or 3′ flanking sequences naturally associated with the FKHL7 gene; (ii)contacting the probe/primer with an appropriate nucleic acid containingsample; and (iii) detecting, by hybridization of the probe/primer to thenucleic acid, the presence or absence of the genetic alterationParticularly preferred embodiments comprise 1) sequencing at least aportion of an FKHL7 gene, 2) performing a single strand conformationpolymorphism (SSCP) analysis to detect differences in electrophoreticmobility between mutant and wild-type nucleic acids; and 3) detecting orquantitating the level of an FKHL7 protein in an immunoassay using anantibody which is specifically immunoreactive with a wild-type ormutated FKHL7 protein.

Information obtained using the diagnostic assays described herein (aloneor in conjunction with information on another genetic defect, whichcontributes to the same disease) is useful for diagnosing or confirmingthat a symptomatic subject has a genetic defect (e.g. in an FKHL7 geneor in a gene that regulates the expression of an FKHL7 gene), whichcauses or contributes to the particular disease or disorder.Alternatively, the information (alone or in conjunction with informationon another genetic defect, which contributes to the same disease) can beused prognostically for predicting whether a non-symptomatic subject islikely to develop a disease or condition, which is caused by orcontributed to by an abnormal FKHL7 activity or protein level in asubject. In particular, the assays permit one to ascertain anindividual's predilection to develop a condition associated with amutation in FKHL7, where the mutation is a single nucleotidepolymorphism (SNP). Based on the prognostic information, a doctor canrecommend a regimen (e.g. diet or exercise) or therapeutic protocoluseful for preventing or prolonging onset of a congenital heart diseasein the individual.

In addition, knowledge of the particular alteration or alterations,resulting in defective or deficient FKHL7 genes or proteins in anindividual, alone or in conjunction with information on other geneticdefects contributing to the same disease (the genetic profile of theparticular disease) allows customization of therapy to the individual'sgenetic profile, the goal of pharmacogenomics. For example, anindividual's FKHL7 genetic profile or the genetic profile of thecongenital heart disease can enable a doctor to: 1) more effectivelyprescribe a drug that will address the molecular basis of glaucoma; and2) better determine the appropriate dosage of a particular drug. Forexample, the expression level of FKHL7 proteins, alone or in conjunctionwith the expression level of other genes known to be involved inglaucoma, can be measured in many patients at various stages of thedisease to generate a transcriptional or expression profile of thecongenital heart disease. Expression patterns of individual patients canthen be compared to the expression profile of the congenital heartdisease to determine the appropriate drug and dose to administer to thepatient.

The ability to target populations expected to show the highest clinicalbenefit, based on the FKHL7 or congenital heart disease genetic profile,can enable: 1) the repositioning of marketed drugs with disappointingmarket results; 2) the rescue of drug candidates whose clinicaldevelopment has been discontinued as a result of safety or efficacylimitations, which are patient subgroup-specific; and 3) an acceleratedand less costly development for drug candidates and more optimal druglabeling (e.g. since the use of FKHL7 as a marker is useful foroptimizing effective dose).

In another aspect, the invention provides methods for identifying acompound which modulates an FKHL7 activity, e.g. the interaction betweenan FKHL7 polypeptide and a target peptide In a preferred embodiment, themethod includes the steps of (a) forming a reaction mixture, whichincludes: (i) an FKHL7 polypeptide, (ii) an FKHL7 binding partner and(iii) a test compound; and (b) detecting interaction of the FKHL7polypeptide and the FKHL7 binding partner. A statistically significantchange (potentiation or inhibition) in the interaction of the FKHL7polypeptide and FKHL7 binding partner in the presence of the testcompound, relative to the interaction in the absence of the testcompound, indicates a potential agonist (mimetic or potentiator) orantagonist (inhibitor) of FKHL7 bioactivity for the test compound. Thereaction mixture can be a cell-free protein preparation, e.g., areconstituted protein mixture or a cell lysate, or it can be arecombinant cell including a heterologous nucleic acid recombinantlyexpressing the FKHL7 binding partner.

In preferred embodiments, the step of detecting interaction of the FKHL7and FKHL7 binding partner is a competitive binding assay. In otherpreferred embodiments, at least one of the FKHL7 polypeptide and theFKHL7 binding partner comprises a detectable label, and interaction ofthe FKHL7 and FKHL7 binding partner is quantified by detecting the labelin the complex. The detectable label can be, e.g., a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. In otherembodiments, the complex is detected by an immunoassay.

Yet another exemplary embodiment provides an assay for screening testcompounds to identify agents which modulate the amount of FKHL7 producedby a cell. In one embodiment, the screening assay comprises contacting acell transfected with a reporter gene operably linked to an FKHL7promoter with a test compound and determining the level of expression ofthe reporter gene. The reporter gene can encode, e.g., a gene productthat gives rise to a detectable signal such as color, fluorescence,luminescence, cell viability, relief of a cell nutritional requirement,cell growth, and drug resistance. For example, the reporter gene canencode a gene product selected from the group consisting ofchloramphenicol acetyl transferase, luciferase, beta-galactosidase andalkaline phosphatase.

Also within the scope of the invention are methods for treating acongenital heart disease, comprising administering (e g., either locallyor systemically) to a subject, a pharmaceutically effective amount of acomposition comprising an FKHL7 therapeutic.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

3. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B are a DNA sequence of human FKHL7 gene including the 5′ and3′ untranslated regions (UTRs) (SEQ ID No. 1). The 1659 base pair openreading frame is provided herein as SEQ ID NO. 3 and the (SEQ ID NO. 3)553 amino acid human FKHL7 protein is provided herein as SEQ ID No. 2.The forkhead region of the protein is indicated by underline.

FIG. 2 shows an amino acid comparison of the forkhead domains ofdifferent members of the FKHL-family of genes (SEQ ID NOS: 4-20). Thelocations of the three alpha helices and the two wing domains are shown(Clark, K. L. et al., Natural 364:412:420 (1993)). The Drosophilaforkhead gene sequence is shown above that for FKHL7, while thepositions of the three missense mutations are shown below FKHL7.Translation of the 11 base pair deletion (bp del) mutation results intotal loss of the forkhead domain. The other FKHL family members areshown below FKHL7 for comparison. For FKHL10, only partial sequence isavailable for forkhead domain. The last sequence shown is that for thedistantly related FKHR which has been mapped to 13Q14 near the R1EG2locus.

FIG. 3 provides the identity the location of Expressed Sequence Tags(ESTs) that map to regions of the human FKHL7 gene.

4. DETAILED DESCRIPTION OF THE INVENTION

4.1. General

The present invention is based, at least in part, on the discovery of anovel human gene, termed “hFKHL7” and the finding that defects in thegene result in the development of congenital heart disease. Moreparticularly, a one base pair deletion upstream of the FKHL7 forkheaddomain, resulting in a truncated protein that lacks the forkhead domainwas found in two individuals from a nuclear family. The proband wasfound to have Rieger anomaly similar to other patients as well as anatrial septal defect. His mother has Rieger anomaly Both individualswere found to harbor this mutation. However, the mutation was not foundin 128 normal Caucasian individuals. In addition, as shown in thefollowing FIG. 3, of the 26 human ESTs identified in a BLASTN search, 5were found to be derived from a fetal heart library and 2 were from anaorta cDNA library. Furthermore, an additional 4 ESTs were from a pooledlibrary of three tissues (melanocytes, uterus and fetal heart). The factthat such a large proportion of the ESTs are derived from heart furthersupports the finding that mutations in the gene can result in congenitalheart deflects. Expression of FKHL7 by Northern blot analysis has beenconfirmed in human and mouse heart.

hFKHL7 maps to human chromosome 6p25. The FKHL7 protein is a monomericDNA binding protein that shares a core binding site (RTAAYA) with fourother FKHL7-like proteins. The human FKHL7 coding sequence is 1.7 kb insize and contains no introns. The 1659 bp open reading frame (SEQ ID NO.3) encodes a 553 amino acid polypeptide (SEQ ID NO. 2). The firstin-frame ATG was found to match well with the Kozak consensus sequence(Kozak, M. Mamm. Genome 7: 5630574 (1996) and Kozak, M. Anna Rev. Cell.Biol. 8: 197-922 (1992)). The COOH-terminal domain contains severalstretches of homopolymeric runs of alanine and glycine The FKHL7 codingregion contains 5 recognition sites for the restriction enzyme NotI. ABLASTN screen of the public dbEST database with the FKHL7 genomicsequence yields only partial human and mouse cDNA coverage of this gene(SEE FIG. 1). Based on the analysis of cDNA clones identified in thepublic databases, there is evidence for the utilization of at least twodifferent polyadenylation signals within the 3′ untranslated region.

Human FKHL7 is most abundantly expressed during embryogenesis and of theadult tissues tested, significant expression was observed in adult eye,heart, kidney and lung, while relatively little to no expression wasobserved in adult skeletal muscle, spleen or liver.

4.2 Definitions

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below.

The term “agonist”, as used herein, is meant to refer to an agent thatmimics or upregulates (e.g. potentiates or supplements) an FKHL7bioactivity. An FKHL7 agonist can be a wild-type FKHL7 protein orderivative thereof having at least one bioactivity of the wild-typeFKHL7. An FKHL7 therapeutic can also be a compound that upregulatesexpression of an FKHL7 gene or which increases at least one bioactivityof an FKHL7 protein. An agonist can also be a compound which increasesthe interaction of an FKHL7 polypeptide with another molecule, e.g. anupstream region of a gene, which is regulated by an FKHL7 transcriptionfactor.

“Antagonist” as used herein is meant to refer to an agent thatdown-regulates (e.g. suppresses or inhibits) at least one FKHL7bioactivity. An FKHL7 antagonist can be a compound which inhibits ordecreases the interaction between an FKHL7 protein and another molecule,e.g, an upstream region of a gene, which is regulated by an FKHL7transcription factor. Accordingly, a preferred antagonist is a compoundwhich inhibits or decreases binding to an upstream region of a gene,which is regulated by an FKHL7 transcription factor and thereby blockssubsequent activation of the FKHL7. An antagonist can also be a compoundthat downregulates expression of an FKHL7 gene or which reduces theamount of FKHL7 protein present. The FKHL7 antagonist can be a dominantnegative form of an FKHL7 polypeptid, e.g., form of an FKHL7 polypeptidewhich is capable of interacting with an upstream region of a gene, whichis regulated by an FKHL7 transcription factor, but which is not capableof regulating transcription The FKHL7 antagonist can also be a nucleicacid encoding a dominant negative form of an FKHL7 polypeptide, an FKHL7antisense nucleic acid, or a ribozyme capable of interactingspecifically with an FKHL7 RNA. Yet other FKHL7 antagonists aremolecules which bind to an FKHL7 polypeptide and inhibit its action.Such molecules include peptides, antibodies and small molecules.

The term “allele”, which is used interchangeably herein with “allelicvariant” refers to alternative forms of a gene or portions thereof.Alleles occupy the same locus or position on homologous chromosomes.When a subject has two identical alleles of a gene, the subject is saidto be homozygous for the gene or allele. When a subject has twodifferent alleles of a gene, the subject is said to be heterozygous forthe gene. Alleles of a specific gene can differ from each other in asingle nucleotide, or several nucleotides, and can includesubstitutions, deletions, and insertions of nucleotides. An allele of agene can also be a form of a gene containing a mutation. The term“allelic variant of a polymorphic region of an FKHL7 gene” refers to aregion of an FKHL7 gene having one or several nucleotide sequences foundin that region of the gene in other individuals.

“Biological activity” or “bioactivity” or “activity” or “biologicalfunction”, which are used interchangeably, for the purposes herein meansan effector or antigenic function that is directly or indirectlyperformed by an FKHL7 polypeptide (whether in its native or denaturedconformation), or by any subsequence thereof. Biological activitiesinclude binding to a target nucleic acid e.g, an upstream region of agene, which is regulated by an FKHL7 transcription factor. An FKHL7bioactivity can be modulated by directly affecting an FKHL7 polypeptide.Alternatively, an FKHL7 bioactivity can be modulated by modulating thelevel of an FKHL7 polypeptide, such as by modulating expression of anFKHL7 gene.

As used herein the term “bioactive fragment of an FKHL7 polypeptide”refers to a fragment of a full-length FKHL7 polypeptide, wherein thefragment specifically mimics or antagonizes the activity of a wild-typeFKHL7 polypeptide. The bioactive fragment preferably is a fragmentcapable of interacting with e.g, an upstream region of a gene, which isregulated by an FKHL7 transcription factor.

The term “an aberrant activity”, as applied to an activity of apolypeptide such as FKHL7, refers to an activity which differs from theactivity of the wild-type or native polypeptide or which differs fromthe activity of the polypeptide in a healthy subject. An activity of apolypeptide can be aberrant because it is stronger than the activity ofits native counterpart Alternatively, an activity can be aberrantbecause it is weaker or absent relative to the activity of its nativecounterpart. An aberrant activity can also be a change in an activity.For example an aberrant polypeptide can interact with a different targetpeptide. A cell can have an aberrant FKHL7 activity due tooverexpression or underexpression of the gene encoding FKHL7.

“Cells”, “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A “chimeric polypeptide” or “fusion polypeptide” is a fusion of a firstamino acid sequence encoding one of the subject FKHL7 polypeptides witha second amino acid sequence defining a domain (e.g. polypeptideportion) foreign to and not substantially homologous with any domain ofan FKHL7 polypeptide. A chimeric polypeptide may present a foreigndomain which is found (albeit in a different polypeptide) in an organismwhich also expresses the first polypeptide, or it may be an“interspecies”, “intergenic”, etc. fusion of polypeptide structuresexpressed by different kinds of organisms. In general, a fusionpolypeptide can be represented by the general formula X-FKHL7-Y, whereinFKHL7 represents a portion of the polypeptide which is derived from anFKHL7 polypeptide, and X and Y are independently absent or representamino acid sequences which are not related to an FKHL7 sequence in anorganism, including naturally occurring mutants.

“Congenital heart disease” refers to defects in the heart and majorgreat vessels produced by abnormalities at various stages of fetaldevelopment and present at birth, but which may not be diagnosed untillater. Examples include: ventricular septal defect, atrial septaldefect, patent ductus arteriosus, atrioventricular canal defects,congential aortic valve stenosis, pulmonic valve stenosis, peripheralpulmonic stenosis, coarctation of the aorta, tetralogy of fallot,transposition of the great arteries, complex cyanotic congenital heartdisease and underdeveloped left ventricle syndrome.

The term “nucleotide sequence complementary to the nucleotide sequenceset forth in SEQ ID NO. x” refers to the nucleotide sequence of thecomplementary strand of a nucleic acid strand having SEQ ID NO x. Theterm “complementary strand” is used herein interchangeably with the term“complement”. The complement of a nucleic acid strand can be thecomplement of a coding strand or the complement of a non-coding strand.When referring to double stranded nucleic acids, the complement of anucleic acid having SEQ ID NO. x refers to the complementary strand ofthe strand having SEQ ID NO. x or to any nucleic acid having thenucleotide sequence of the complementary strand of SEQ ID NO. x. Whenreferring to a single stranded nucleic acid having the nucleotidesequence SEQ ID NO. x, the complement of this nucleic acid is a nucleicacid having a nucleotide sequence which is complementary to that of SEQID NO. x. The nucleotide sequences and complementary sequences thereofare always given in the 5′ to 3′ direction.

A “delivery complex” shall mean a targeting means (e.g. a molecule thatresults in higher affinity binding of a gene, protein, polypeptide orpeptide to a target cell surface and/or increased cellular or nuclearuptake by a target cell). Examples of targeting means include: sterols(e.g. cholesterol), lipids (e.g. a cationic lipid, virosome orliposome), viruses (e.g. adenovirus, adeno-associated virus, andretrovirus) or target cell specific binding agents (e.g. ligandsrecognized by target cell specific receptors). Preferred complexes aresufficiently stable in vivo to prevent significant uncoupling prior tointernalization by the target cell. However, the complex is cleavableunder appropriate conditions within the cell so that the gene, protein,polypeptide or peptide is released in a functional form.

As is well known, genes may exist in single or multiple copies withinthe genome of an individual. Such duplicate genes may be identical ormay have certain modifications, including nucleotide substitutions,additions or deletions, which all still code for polypeptides havingsubstantially the same activity. The term “DNA sequence encoding anFKHL7 polypeptide” may thus refer to one or more genes within aparticular individual. Moreover, certain differences in nucleotidesequences may exist between individual organisms, which are calledalleles. Such allelic differences may or may not result in differencesin amino acid sequence of the encoded polypeptide, yet still encode apolypeptide with the same biological activity

The term “FKHL7 nucleic acid” refers to a nucleic acid encoding an FKHL7protein, such as nucleic acids having SEQ ID NOs. 1 or 3, as well asfragments thereof, complements thereof, and derivatives thereof.

The terms “FKHL7 polypeptide” and “FKHL7 protein” are intended toencompass polypeptides comprising the amino acid sequence shown as SEQID NO. 2 or fragments thereof, and homologs thereof and include agonistand antagonist polypeptides.

The term “FKHL7 therapeutic” refers to various forms of FKHL7polypeptides, as well as peptidomimetics, nucleic acids, or smallmolecules, which can modulate at least one activity of an FKHL7polypeptide, e.g., binding to and/or otherwise regulating expression ofa gene, by mimicking or potentiating (agonizing) or inhibiting(antagonizing) the effects of a naturally-occurring FKHL7 polypeptide.An FKHL7 therapeutic which mimics or potentiates the activity of awild-type FKHL7 polypeptide is a “FKHL7 agonist”. Conversely, an FKHL7therapeutic which inhibits the activity of a wild-type FKHL7 polypeptideis a “FKHL7 antagonist”.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare identical at that position. A degree of homology or similarity oridentity between nucleic acid sequences is a function of the number ofidentical or matching nucleotides at positions shared by the nucleicacid sequences. An “unrelated” or “non-homologous” sequence shares lessthan about 40% identity, though preferably less than about 25% identity,with one of the FKHL7 sequences of the present invention.

The term “interact” as used herein is meant to include detectablerelationships or associations (e.g. biochemical interactions) betweenmolecules, such as interaction between protein-protein, protein-nucleicacid, nucleic acid-nucleic acid, and protein-small molecule or nucleicacid-small molecule in nature.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs, or RNAs,respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding one of thesubject FKHL7 polypeptides preferably includes no more than about 10kilobases (kb) of nucleic acid sequence which naturally immediatelyflanks the FKHL7 gene in genomic DNA, more preferably no more than about5 kb of such naturally occurring flanking sequences, and most preferablyless than about 1.5 kb of such naturally occurring flanking sequence.The term isolated as used herein also refers to a nucleic acid orpeptide that is substantially free of cellular material, viral material,or culture medium when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized.Moreover, an “isolated nucleic acid” is meant to include nucleic acidfragments which are not naturally occurring as fragments and would notbe found in the natural state. The term “isolated” is also used hereinto refer to polypeptides which are isolated from other cellular proteinsand is meant to encompass both purified and recombinant polypeptides.

The term “modulation” as used herein refers to both upregulation (i.e.,activation or stimulation (e.g., by agonizing or potentiating)) anddownregulation (i.e. inhibition or suppression (e.g., by antagonizing,decreasing or inhibiting)).

The term “mutated gene” refers to an allelic form of a gene, which iscapable of altering the phenotype of a subject having the mutated generelative to a subject which does not have the mutated gene. If a subjectmust be homozygous for this mutation to have an altered phenotype, themutation is said to be recessive. If one copy of the mutated gene issufficient to alter the genotype of the subject, the mutation is said tobe dominant. If a subject has one copy of the mutated gene and has aphenotype that is intermediate between that of a homozygous and that ofa heterozygous subject (for that gene), the mutation is said to beco-dominant.

The “non-human animals” of the invention include mammals such asrodents, non-human primates, sheep, dog, cow, chickens, amphibians,reptiles, etc. Preferred non-human animals are selected from the rodentfamily including rat and mouse, most preferably mouse, though transgenicamphibians, such as members of the Xenopus genus, and transgenicchickens can also provide important tools for understanding andidentifying agents which can affect, for example, embryogenesis andtissue formation. The term “chimeric animal” is used herein to refer toanimals in which the recombinant gene is found, or in which therecombinant gene is expressed in some but not all cells of the animal.The term “tissue-specific chimeric animal” indicates that one of therecombinant FKHL7 genes is present and/or expressed or disrupted in sometissues but not others.

As used herein, the term “nucleic acid” refers to polynucleotides oroligonucleotides such as deoxyribonucleic acid (DNA), and, whereappropriate, ribonucleic acid (RNA). The term should also be understoodto include, as equivalents, analogs of either RNA or DNA made fromnucleotide analogs and as applicable to the embodiment being described,single (sense or antisense) and double-stranded polynucleotides.

The term “polymorphism” refers to the coexistence of more than one formof a gene or portion (e.g., allelic variant) thereof A portion of a geneof which there are at least two different forms, i.e., two differentnucleotide sequences, is referred to as a “polymorphic region of agene”. A polymorphic region can be a single nucleotide, the identity ofwhich differs in different alleles. A polymorphic region can also beseveral nucleotides long.

A “polymorphic gene” refers to a gene having at least one polymorphicregion.

As used herein, the term “promoter” refers to a DNA sequence thatregulates expression of a selected DNA sequence operably linked to thepromoter, and which effects expression of the selected DNA sequence incells. The term encompasses “tissue specific” promoters, i.e. promoters,which effect expression of the selected DNA sequence only in specificcells (e.g. cells of a specific tissue). The term also covers so-called“leaky” promoters, which regulate expression of a selected DNA primarilyin one tissue, but cause expression in other tissues as well The termalso encompasses non-tissue specific promoters and promoters thatconstitutively express or that are inducible (i.e. expression levels canbe controlled).

The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein when referring to a gene product.

The term “recombinant protein” refers to a polypeptide of the presentinvention which is produced by recombinant DNA techniques, whereingenerally, DNA encoding an FKHL7 polypeptide is inserted into a suitableexpression vector which is in turn used to transform a host cell toproduce the heterologous protein. Moreover, the phrase “derived from”,with respect to a recombinant FKHL7 gene, is meant to include within themeaning of “recombinant protein” those proteins having an amino acidsequence of a native FKHL7 polypeptide, or an amino acid sequencesimilar thereto which is generated by mutations including substitutionsand deletions (including truncation) of a naturally occurring form ofthe polypeptide.

“Small molecule” as used herein, is meant to refer to a composition,which has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be nucleic acids, peptides,polypeptides, peptidomimetics, carbohydrates, lipids or other organic(carbon containing) or inorganic molecules. Many pharmaceuticalcompanies have extensive libraries of chemical and/or biologicalmixtures, often fungal, bacterial, or algal extracts, which can bescreened with any of the assays of the invention to identify compoundsthat modulate an FKHL7 bio-activity.

As used herein, the term “specifically hybridizes” or “specificallydetects” refers to the ability of a nucleic acid molecule of theinvention to hybridize to at least approximately 6, 12, 20, 30, 50, 100,150, 200, 300, 350, 400, 425, 450, 475 or 500 consecutive nucleotides ofa vertebrate gene, preferably an FKHL7 gene.

“Transcriptional regulatory sequence” is a generic term used throughoutthe specification to refer to DNA sequences, such as initiation signals,enhancers, and promoters, which induce or control transcription ofprotein coding sequences with which they are operably linked. Inpreferred embodiments, transcription of one of the FKHL7 genes is underthe control of a promoter sequence (or other transcriptional regulatorysequence) which controls the expression of the recombinant gene in acell-type in which expression is intended. It will also be understoodthat the recombinant gene can be under the control of transcriptionalregulatory sequences which are the same or which are different fromthose sequences which control transcription of the naturally-occurringforms of a FKHL7 polypeptide.

As used herein, the term “transfection” means the introduction of anucleic acid, e.g., via an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of an FKHL7 polypeptideor, in the case of anti-sense expression from the transferred gene, theexpression of a naturally-occurring form of the FKHL7 polypeptide isdisrupted.

As used herein, the term “transgene” means a nucleic acid sequence(encoding, e.g., one of the FKHL7 polypeptides, or an antisensetranscript thereto) which has been introduced into a cell. A transgenecould be partly or entirely heterologous, i.e., foreign, to thetransgenic animal or cell into which it is introduced, or, can behomologous to an endogenous gene of the transgenic animal or cell intowhich it is introduced, but which is designed to be inserted, or isinserted, into the animal's genome in such a way as to alter the genomeof the cell into which it is inserted (e.g., it is inserted at alocation which differs from that of the natural gene or its insertionresults in a knockout). A transgene can also be present in a cell in theform of an episome. A transgene can include one or more transcriptionalregulatory sequences and any other nucleic acid, such as introns, thatmay be necessary for optimal expression of a selected nucleic acid.

A “transgenic animal” refers to any animal, preferably a non-humanmammal, bird or an amphibian, in which one or more of the cells of theanimal contain heterologous nucleic acid introduced by way of humanintervention, such as by transgenic techniques well known in the art.The nucleic acid is introduced into the cell, directly or indirectly byintroduction into a precursor of the cell, by way of deliberate geneticmanipulation, such as by microinjection or by infection with arecombinant virus. The term genetic manipulation does not includeclassical cross-breeding, or in vitro fertilization, but rather isdirected to the introduction of a recombinant DNA molecule. Thismolecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a recombinantform of one of the FKHL7 polypeptides, e.g. either agonistic orantagonistic forms. However, transgenic animals in % which therecombinant FKHL7 gene is silent are also contemplated, as for example,the FLP or CRE recombinase dependent constructs described below.Moreover, “transgenic animal” also includes those recombinant animals inwhich gene disruption of one or more FKHL7 genes is caused by humanintervention, including both recombination and antisense techniques.

The term “treating” as used herein is intended to encompass curing aswell as ameliorating at least one symptom of the condition or disease.

The term “vector” refers to a nucleic acid molecule, which is capable oftransporting another nucleic acid to which it has been linked. One typeof preferred vector is an episome, i.e., a nucleic acid capable ofextra-chromosomal replication. Preferred vectors are those capable ofautonomous replication and/or expression of nucleic acids to which theyare linked. Vectors capable of directing the expression of genes towhich they are operatively linked are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of “plasmids” which refer generally tocircular double stranded DNA loops which, in their vector form are notbound to the chromosome. In the present specification, “plasmid” and“vector” are used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors which serve equivalent functions andwhich become known in the art subsequently hereto.

The term “wild-type allele” refers to an allele of a gene which, whenpresent in two copies in a subject results in a wild-type phenotype.There can be several different wild-type alleles of a specific gene,since certain nucleotide changes in a gene may not affect the phenotypeof a subject having two copies of the gene with the nucleotide changes.

4.3. Nucleic Acids of the Present Invention

The invention provides FKHL7 nucleic acids, homologs thereof, andportions thereof. Preferred nucleic acids have a sequence, which is atleast about 60%, 61%, 62%, 63%, 64% o, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, and more preferably85% homologous with a nucleotide sequence of an FKHL7 gene, e.g., suchas a sequence shown in one of SEQ ID NOS: 1 or 3 or complements thereof.Nucleic acids at least 90%, more preferably 95%, and most preferably atleast about 98-99% homologous with a nucleic sequence represented in oneof SEQ ID NOS. 1 or 3 or a complement thereof are of course also withinthe scope of the invention.

The invention also pertains to isolated nucleic acids comprising anucleotide sequence encoding FKHL7 polypeptides, variants and/orequivalents of such nucleic acids. The term equivalent is understood toinclude nucleotide sequences encoding functionally equivalent FKHL7polypeptides or functionally equivalent peptides having an activity ofan FKHL7 protein such as described herein. Equivalent nucleotidesequences will include sequences that differ by one or more nucleotidesubstitutions, additions or deletions, such as allelic variants, andtherefore includes sequences that differ from the nucleotide sequence ofthe FKHL7 gene shown in SEQ ID NOS. 1 or 3, due to the degeneracy of thegenetic code.

Preferred nucleic acids are vertebrate FKHL7 nucleic acids. Particularlypreferred vertebrate FKHL7 nucleic acids are mammalian. Regardless ofspecies, particularly preferred FKHL7 nucleic acids encode polypeptidesthat are at least about 60%, 65%, 70/o, 72/o, 74% o, 76%, 78/o, 80% o,90%, or 95% similar or identical to an amino acid sequence of avertebrate FKHL7 protein. In one embodiment, the nucleic acid is a cDNAencoding a polypeptide having at east one bio-activity of the subjectFKHL7 polypeptide. Preferably, the nucleic acid includes all or aportion of the nucleotide sequence corresponding to the nucleic acid ofSEQ ID NOS. 1 or 3.

Still other preferred nucleic acids of the present invention encode anFKHL7 polypeptide which is comprised of at least 50, 100, 150, 200, 250,300, 350, 400, 450 or 500 amino acid residues. For example, such nucleicacids can comprise about 150, 300, 450, 600, 750, 900, 1050, 1200, 135(1or 1500 base pairs. Also within the scope of the invention are nucleicacid molecules for use as probes/primer or antisense molecules (i.e.non-coding nucleic acid molecules), which can comprise at least about 6,12, 20, 30, 50, 60, 70, 80, 90 or 100 base pairs in length

Another aspect of the invention provides a nucleic acid which hybridizesunder stringent conditions to a nucleic acid represented by SEQ ID NOS.1 or 3 or a complement thereof. Appropriate stringency conditions whichpromote DNA hybridization, for example, 6.0× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C.,are known to those skilled in the art or can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. For example, the salt concentration in the wash step can beselected from a low stringency of about 2.0×SSC at 50° C. to a highstringency of about 0.2×SSC at 50° C. In addition, the temperature inthe wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or temperature and saltconcentration may be held constant while the other variable is changed.In a preferred embodiment, an FKHL7 nucleic acid of the presentinvention will bind to one of SEQ ID NOS. 1 or 3 or complement thereofunder moderately stringent conditions, for example at about 2.0×SSC andabout 40° C. In a particularly preferred embodiment, an FKHL7 nucleicacid of the present invention will bind to one of SEQ ID NOS. 1 or 3 ora complement thereof under high stringency conditions.

Nucleic acids having a sequence that differs from the nucleotidesequences shown in one of SEQ ID NOS. 1 or 3 or a complement thereof dueto degeneracy in the genetic code are also within the scope of theinvention. Such nucleic acids encode functionally equivalent peptides(i.e., peptides having a biological activity of an FKHL7 polypeptide)but differ in sequence from the sequence shown in the sequence listingdue to degeneracy in the genetic code. For example, a number of aminoacids are designated by more than one triplet. Codons that specify thesame amino acid, or synonyms (for example, CAU and CAC each encodehistidine) may result in “silent” mutations which do not affect theamino acid sequence of an FKHL7 polypeptide. However, it is expectedthat DNA sequence polymorphisms that do lead to changes in the aminoacid sequences of the subject FKHL7 polypeptides will exist amongmammals. One skilled in the art will appreciate that these variations inone or more nucleotides (e.g., up to about 3-5% of the nucleotides) ofthe nucleic acids encoding polypeptides having an activity of an FKHL7polypeptide may exist among individuals of a given species due tonatural allelic variation.

The polynucleotide of the present invention may also be fused in frameto a man ker sequence, also referred to herein as “Tag sequence”encoding a “Tag peptide”, which allows for marking and/or purificationof the polypeptide of the present invention. In a preferred embodiment,the marker sequence is a hexahistidine tag, e.g., supplied by a PQE-9vector. Numerous other lag peptides are available commercially. Otherfrequently used Tags include myc-epitopes (e.g., see Ellison et al. (I991) J Biol Chem 266:21150-21157) which includes a 10-residue sequencefrom c-myc, the pFLAG system (International Biotechnologies, Inc.), thepEZZ-protein A system (Pharmacia, N.J.), and a 16 amino acid portion ofthe Haemophilus influenza hemagglutinin protein. Furthermore, anypolypeptide can be used as a Tag so long as a reagent, e.g., an antibodyinteracting specifically with the Tag polypeptide is available or can beprepared or identified.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant protein, canallow purification of the expressed fusion protein by affinitychromatography using a Ni²⁺ metal resin. The purification leadersequence can then be subsequently removed by treatment with enterokinaseto provide the purified protein (e.g., see Hochuli et al. (1987) J.Chromatography 411:177, and Janknecht et al. PNAS 88:8972).

Techniques for making fusion genes are known to those skilled in theart. Essentially, the joining of various DNA fragments coding fordifferent polypeptide sequences is performed in accordance withconventional techniques, employing blunt-ended or stagger-ended terminifor ligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

Other preferred FKHL7 fusion proteins include FKHL7-immunoglobulin(FKHL7-1 g) polypeptides. An FKHL7-1 g polypeptide can comprise theentire extracellular domain of FKHL7, e.g. human FKHL7, or a variantthereof. For example, an FKHL7-1 g fusion proteins can be prepared asdescribed e.g., in U.S. Pat. No. 5,434,131.

As indicated by the examples set out below, FKHL7 protein-encodingnucleic acids can be obtained from mRNA, present in any of a number ofeukaryotic cells, e.g. from cardiac tissue. It should also be possibleto obtain nucleic acids encoding FKHL7 polypeptides of the presentinvention from genomic DNA from both adults and embryos. For example, agene encoding an FKHL7 protein can be cloned from either a cDNA or agenomic library in accordance with protocols described herein, as wellas those generally known to persons skilled in the art. cDNA encoding anFKHL7 protein can be obtained by isolating total mRNA from a cell, erg.,a vertebrate cell, a mammalian cell, or a human cell, includingembryonic cells. Double stranded cDNAs can then be prepared from thetotal mRNA and subsequently inserted into a suitable plasmid orbacteriophage vector using any one of a number of known techniques. Thegene encoding an FKHL7 protein can also be cloned using establishedpolymerase chain reaction techniques in accordance with the nucleotidesequence information provided by the invention. The nucleic acid of theinvention can be DNA or RNA or analogs thereof A preferred nucleic acidis a cDNA represented by a sequence selected from the group consistingof SEQ ID NOS. 1 or 3.

Preferred nucleic acids encode a vertebrate FKHL7 polypeptide comprisingan amino acid sequence that is at least about 60% homologous, morepreferably at least about 70% homologous and most preferably at leastabout 80% homologous with an amino acid sequence contained in SEQ ID NO.2. Nucleic acids which encode polypeptides with at least about 90%, morepreferably at least about 95%, and most preferably at least about 98-99%homology with an amino acid sequence represented in SEQ ID NO. 2 arealso within the scope of the invention. In one embodiment, the nucleicacid is a cDNA encoding, a peptide having at least one activity of thesubject vertebrate FKHL7 polypeptide. Preferably, the nucleic acidincludes all or a portion of the nucleotide sequence corresponding tothe coding region of SEQ ID NOS.

Preferred nucleic acids encode a bioactive fragment of a vertebrateFKHL7 polypeptide comprising an amino acid sequence, which is at leastabout 60% homologous or identical, more preferably at least about 70%homologous or identical, still more preferably at least about 75%homologous or identical and most preferably at least about 80%homologous or identical with an amino acid sequence of SEQ ID NO. 2.Nucleic acids which encode polypeptides which are at least about 90%,more preferably at least about 95%, and most preferably at least about98-99% homologous or identical, with in amino acid sequence representedin SEQ ID NO. 2 are also within the scope of the invention.

Bioactive fragments of FKHL7 polypeptides can be polypeptides, whichbind upstream of and/or regulate the expression of a genie Assays fordetermining whether an FKHL117 polypeptide has any of these or otherbiological activities are known in the art and are further describedherein

Nucleic acids encoding modified forms or mutant forms of FKHL7 alsoinclude those encoding FKHL7 proteins having mutated glycosylationsites, such that either the encoded FKHL7 protein is not glycosylated,partially glycosylated and/or has a modified glycosylation pattern.

Other preferred nucleic acids of the invention include nucleic acidsencoding derivatives of FKHL7 polypeptides which lack one or morebiological activities of FKHL7 polypeptides. Such nucleic acids can beobtained, e.g., by a first round of screening of libraries for thepresence or absence of a first activity and a second round of screeningfor the presence or absence of another activity.

Also within the scope of the invention are nucleic acids encoding splicevariants or nucleic acids representing transcripts synthesized from analternative transcriptional initiation site, such as those whosetranscription was initiated from a site in an intron.

In preferred embodiments, the FKHL7 nucleic acids can be modified at thebase moiety, sugar moiety or phosphate backbone to improve, e.g., thestability, hybridization, or solubility of the molecule. For example,the deoxyribose phosphate backbone of the nucleic acids can be modifiedto generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic& Medical Chemistry 4 (1): 5-23). As used herein, the terms “peptidenucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics,in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs has been shown to allow forspecific hybridization to DNA and RNA under conditions of low ionicstrength. The synthesis of PNA oligomers can be performed using standardsolid phase peptide synthesis protocols as described in Hyrup B. et al.(1996) supra, Perry-O'Keefe et al. PNAS 93: 14670-675.

PNAs of FKHL7 can be used in therapeutic and diagnostic applications andare further described herein. Such modified nucleic acids can be used asantisense or antigene agents for sequence-specific modulation of geneexpression or in the analysis of single base pair mutations in a geneby, e.g., PNA directed PCR clamping or as probes or primers for DNAsequence and hybridization (Hyrup B. et al (1996) supra Perry-O'Keefesupra).

PNAs of FKHL11.7 can further be modified, e.g., to enhance theirstability or cellular uptake, e.g., by attaching lipophilic or otherhelper groups to the FKHL7 PNA, by the formation of PNA-DNA chimeras, orby the use of liposomes or other techniques of drug delivery known inthe art FKHL7 PNAs can also be linked to DNA as described, e.g., inHyrup B. (1996) supra and Finn P. J. et al. (1996) Nucleic AcidsResearch 24 (17): 3357-63. For example, a DNA chain can be synthesizedon a solid support using standard phosphoramidite coupling chemistry andmodified nucleoside analogs, e.g.,5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can beused between the PNA and the 5′ end of DNA (Mag, M. et al. (1989)Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5'PNA segment anda 3′ DNA segment (Finn P. J. et al. (1996) supra). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment (Peterser, K. H. et al. (1975) Bioorganic Med Chem. Lett. 5:1119-11124).

In other embodiments, FKHL7 nucleic acids may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents that facilitate transport across the cell membrane asdescribed herein.

4.3.1 Probes and Primers

The nucleotide sequences determined from the cloning of FKHL7 genes frommammalian organisms % mill further allow for the generation of probesand primers designed for use in identifying and/or cloning FKHL7homologs in other cell types, e.g., from other tissues, as well as FKHL7homologs from other mammalian organisms. For instance, the presentinvention also provides a probe/primer comprising a substantiallypurified oligonucleotide, which oligonucleotide comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast approximately 12, preferably 25, more preferably 40, 50 or 75consecutive nucleotides of sense or anti-sense sequence selected fromthe group consisting of SEQ ID NOS. 1 and 3 or naturally occurringmutants thereof. For instance, primers based on the nucleic acidrepresented in SEQ ID NOS. 1 or 3 can be used in PCR reactions to cloneFKHL7 homologs.

Likewise, probes based on the subject FKHL7 sequences can be used todetect transcripts or genomic sequences encoding the same or homologousproteins, for use, e.g, in prognostic or diagnostic assays (furtherdescribed below). In preferred embodiments, the probe further comprisesa label group attached thereto and able to be detected. e.g., the labelgroup is selected from amongst radioisotopes, fluorescent compoundenzymes, and enzyme co-factors

Probes and primers can be prepared and modified, e.g. as previouslydescribed herein for other types of nucleic acids.

4.3.2 Antisense. Ribozyme and Triplex Techniques

Another aspect of the invention relates to the use of the isolatednucleic acid in “antisense” therapy. As used herein, “antisense” therapyrefers to administration or in situ generation of oligonucleotidemolecules or their derivatives which specifically hybridize (e.g., bind)under cellular conditions, with the cellular mRNA and/or genomic DNAencoding one or more of the subject FKHL7 proteins so as to inhibitexpression of that protein, e.g., by inhibiting transcription and/ortranslation. The binding may be by conventional base paircomplementarity, or, for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix. In general, “antisense” therapy refers to the range oftechniques generally employed in the art, and includes any therapy whichrelies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid which, when transcribed in the cell,produces RNA which is complementary to at least a unique portion of thecellular mRNA which encodes an FKHL7 protein. Alternatively, theantisense construct is an oligonucleotide probe which is generated exvivo and which, when introduced into the cell causes inhibition ofexpression by hybridizing with the mRNA and/or genomic sequences of anFKHL7 gene. Such oligonucleotide probes are preferably modifiedoligonucleotides which are resistant to endogenous nucleases, e.g.,exonucleases and/or endonucleases, and are therefore stable in vitro.Exemplary nucleic acid molecules for use as antisense oligonucleotidesare phosphoramidate, phosphothioate and methylphosphonate analogs of DNA(see also U.S. Pat. Nos. 5,176,996; 5,264,564, and 5,256,775).Additionally, general approaches to constructing oligomers useful inantisense therapy have been reviewed, for example, by Van der Krol etal. (1988) Biotechniques 6:958-976, and Stein et al. (1988) Cancer Res48:2659-2668. With respect to antisense DNA, oligodeoxyribonucleotidesderived from the translation initiation site, e.g., between the −10 and+10 regions of the FKHL7 nucleotide sequence of interest, are preferred.

Antisense approaches involve the design of oligonucleotides (either DNAor RNA) that are complementary to FKHL7 mRNA. The antisenseoligonucleotides will bind to the FKHL7 mRNA transcripts and preventtranslation. Absolute complementarity, although preferred, is notrequired. In the case of double-stranded antisense nucleic acids, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acid.Generally, the longer the hybridizing nucleic acid, the more basemismatches with an RNA it may contain and still form a stable duplex (ortriplex, as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the mRNA, e.g.,the 5′ untranslated sequence up to and including the AUG initiationcodon, should work most efficiently at inhibiting translation. However,sequences complementary to the 3′ untranslated sequences of mRNAs haverecently been shown to be effective at inhibiting translation of mRNAsas well. (Wagner, R. 1994. Nature 372:333). Therefore, oligonucleotidescomplementary to either the 5′ or 3′ untranslated, non-coding regions ofan FKHL7 gene could be used in an antisense approach to inhibittranslation of endogenous FKHL7 mRNA. Oligonucleotides complementary tothe 5′ untranslated region of the mRNA should include the complement ofthe AUG start codon. Antisense oligonucleotides complementary to mRNAcoding regions are less efficient inhibitors of translation but couldalso be used in accordance with the invention. Whether designed tohybridize to the 5′, 3′ or coding region of FKHL7 mRNA, antisensenucleic acids should be at least six nucleotides in length, and arepreferably less than about 100 and more preferably less than about 50,25, 17 or 10 nucleotides in length.

Regardless of the choice of target sequence, it is preferred that iiivitro studies are first performed to quantitate the ability of theantisense oligonucleotide to inhibit gene expression. It is preferredthat these studies utilize controls that distinguish between antisensegene inhibition and nonspecific biological effects of oligonucleotides.It is also preferred that these studies compare levels of the target RNAor protein with that of an internal control RNA or protein.Additionally, it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors), or agents facilitating transport across the cell membrane(see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A.86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652;PCT Publication No. WO88/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalatingagents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including but not limited to5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxyliethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methlylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), %wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

The antisense oligonucleotide can also contain a neutral peptide-likebackbone. Such molecules are termed peptide nucleic acid (PNA)-oligomersand are described, e.g., in Perry-O'Keefe et al. (1996) Proc. Natl.Acad. Sci. U.S.A 93:14670 and in Eglom et al (1993) Nature 365:566 Oneadvantage of PNA oligomers is their ability to bind to complementary DNAessentially independently from the ionic strength of the medium due tothe neutral backbone of the DNA. In yet another embodiment, theantisense oligonucleotide comprises at least one modified phosphatebackbone selected from the group consisting of a phosphorothioate, 3phosphorodithioate, a phosphoamidothioate, a phosphoramidate, aphosphorodiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

In yet a further embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate olgonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc

While antisense nucleotides complementary to the FKHL7 coding regionsequence can be used, those complementary to the transcribeduntranslated region and to the region comprising the initiatingmethionine are most preferred.

The antisense molecules can be delivered to cells which express FKHL7 invivo. A number of methods have been developed for delivering antisenseDNA or RNA to cells, e.g., antisense molecules can be injected directlyinto the tissue site, or modified antisense molecules, designed totarget the desired cells (e.g., antisense linked to peptides orantibodies that specifically bind receptors or antigens expressed on thetarget cell surface) can be administered systematically.

However, it may be difficult to achieve intracellular concentrations ofthe antisense sufficient to suppress translation on endogenous mRNAs incertain instances. Therefore a preferred approach utilizes a recombinantDNA construct in which the antisense oligonucleotide is placed under thecontrol of a strong pol III or pol II promoter. The use of such aconstruct to transfect target cells in the patient will result in thetranscription of sufficient amounts of single stranded RNAs that willform complementary base pairs with the endogenous FKHL7 transcripts andthereby prevent translation of the FKHL7 mRNA. For example, a vector canbe introduced in vivo such that it is taken up by a cell and detects thetranscription of an antisense RNA. Such a vector call remain episomal orbecome chromosomally integrated, as long as it can be transcribed toproduce the desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art, used for replication andexpression in mammalian cells. Expression of the sequence encoding theantisense RNA can be by any promoter known in the art to act inmammalian, preferably human cells. Such promoters can be inducible orconstitutive and can include but not be limited to: the SV40 earlypromoter region (Bernoist and Chambon, 1981, Nature 290:304-310), thepromoter contained in the 3′ long terminal repeat of Rous sarcoma virus(Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinasepromoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al, 1982, Nature 296:39-42), etc Any type of plasmid,cosmid, YAC or viral vector can be used to prepare the recombinant DNAconstruct which can be introduced directly into the tissue site.Alternatively, viral vectors can be used which selectively infect thedesired tissue, in which case administration may be accomplished byanother route (e.g., systematically).

Ribozyme molecules designed to catalytically cleave FKHL7 mRNAtranscripts can also be used to prevent translation of FKHL7 mRNA andexpression of FKHL7 (See, e.g., PCT International PublicationWO90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science247:1222-1225 and U.S. Pat. No. 5,093,246). While ribozymes that cleavemRNA at site specific recognition sequences can be used to destroy FKHL7mRNAs, the use of hammerhead ribozymes is preferred. Hammerheadribozymes cleave mRNAs at locations dictated by flanking regions thatform complementary base pairs with the target mRNA. The sole requirementis that the target mRNA have the following sequence of two bases:5′-UG-3′. The construction and production of hammerhead ribozymes iswell known in the art and is described more fully in Hascloff andGerlach, 1988, Nature, 334:585-591. There are a number of potentialhammerhead ribozyme cleavage sites within the nucleotide sequence ofhuman FKHL7 cDNA Preferably the ribozyme is engineered so that thecleavage recognition site is located near the 5′ end of the FKHL7 mRNA;i.e., to increase efficiency and minimize the intracellular accumulationof non-functional mRNA transcripts

The ribozymes of the present invention can also include RNAendoribonucleases herein after “(ech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena thermophila (known as the IVS, orL.−19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al., 1984, Science, 224;574-579, Zaug andCech, 1986, Science, 231 470-475, Zaug, et al, 1986, Nature,324:429-433; published International patent application No. WO88/04300by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). TheCech-type ribozymes have an eight base pair active site which hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes which targeteight base-pair active site sequences that are present in an FKHL7 gene.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g., for improved stability, targeting, etc.) andshould be delivered to cells which express the FKHL7 gene int vivo. Apreferred method of delivery involves using a DNA construct “encoding”the ribozyme under the control of a strong constitutive pol III or polII promoter, so that transfected cells will produce sufficientquantities of the ribozyme to destroy endogenous FKHL7 messages andinhibit translation. Because ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

Endogenous FKHL7 gene expression can also be reduced by inactivating or“knocking out” the FKHL7 gene or its promoter using targeted homologousrecombination. (e.g., see Smithies et al., 1985, Nature 317:230-234;Thomas & Capecchi, 1987, Cell 51:503-512, Thompson et al., 1989 Cell5:313-321; each of which is incorporated by reference herein in itsentirety). For example, a mutant, non-functional FKHL7 (or a completelyunrelated DNA sequence) flanked by DNA homologous to the endogenousFKHL7 gene (either the coding regions or regulatory regions of the FKHL7gene) can be used, with or without a selectable marker and/or a negativeselectable marker, to transfect cells that express FKHL7 in vivo.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the FKHL7 gene. Such approaches areparticularly suited in the agricultural field where modifications to ES(embryonic stem) cells can be used to generate animal offspring with aninactive FKHL7 (e g., see Thomas & Capecchi 1987 and Thompson 1989,supra). However this approach can be adapted for use in humans providedthe recombinant DNA constructs are directly administered or targeted tothe required site in vivo using appropriate viral vectors.

Alternatively, endogenous FKHL7 gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the FKHL7 gene (i.e., the FKHL7 promoter and/or enhancers) toform triple helical structures that prevent transcription of the FKHL7gene in target cells in the body. (See generally, Helene, C. 1991,Anticancer Drug Des., 6(6):569-84; Helene, C., et al., 1992, Ann. N.Y.Acad. Sci., 660:27-36; and Maher, L. J., 1992, Bioassays 14(12):807-15).

Nucleic acid molecules to be used in triple helix formation for theinhibition of transcription are preferably single stranded and composedof deoxyribonucleotides. The base composition of these oligonucleotidesshould promote triple helix formation via Hoogsteen base pairing rules,which generally require sizable stretches of either purines orpyrimidines to be present on one strand of a duplex. Nucleotidesequences may be pyrimidine-based, which will result in TAT and CGCtriplets across the three associated strands of the resulting triplehelix. The pyrimidine-rich molecules provide base complementarity to apurine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in CGCtriplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′, 3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

Antisense RNA and DNA, ribozyme, and triple helix molecules of theinvention may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors which incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promotersAlternatively, antisense cDNA constricts that synthesize antisense RNAconstitutively or inducibility., depending on the promoter used, can beintroduced stably into cell lines.

Moreover various well known modifications to nucleic acid molecules maybe introduced as a means of increasing, intracellular stability andhalf-life. Possible modifications include but are not limited to theaddition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule or the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages within the oligodeoxyribonucleotide backbone.

4.3.3. Vectors Encoding FKHL7 Proteins and FKHL7 Expressing Cells

The invention further provides plasmids and vectors encoding an FKHL7protein, which can be used to express an FKHL7 protein in a host cell.The host cell may be any prokaryotic or eukaryotic cell Thus, anucleotide sequence derived from the cloning of mammalian FKHL7proteins, encoding all or a selected portion of the full-length protein,can be used to produce a recombinant form of an FKHL7 polypeptide viamicrobial or eukaryotic cellular processes. Ligating the polynucleotidesequence into a gene construct, such as an expression vector, andtransforming or transfecting into hosts, either eukaryotic (yeast,avian, insect or mammalian) or prokaryotic (bacterial) cells, arestandard procedures well known in the art.

Vectors that allow expression of a nucleic acid in a cell are referredto as expression vectors. Typically, expression vectors used forexpressing an FKHL7 protein contain a nucleic acid encoding an FKHL7polypeptide, operably linked to at least one transcriptional regulatorysequence Regulatory sequences are art-recognized and are selected todirect expression of the subject FKHL7 proteins Transcriptionalregulatory sequences are described in Goeddel, Gene ExpressionTechnology. Methods in Enzymology 185, Academic Press. San Diego, Calif.(1990). In one embodiment, the expression vector includes a recombinantgene encoding a peptide having an agonistic activity of a subject FKHL7polypeptide, or alternatively, encoding a peptide which is anantagonistic form of an FKHL7 protein.

Suitable vectors for the expression of an FKHL7 polypeptide includeplasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids,pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmidsfor expression in prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevasiae (see, for example. Broach et al (1983) inexperimental Manipulation of Gene Expression, ed M. Inouye AcademicPress p 83, incorporated by reference herein). These vectors canreplicate in E. coli due the presence of the pBR312 ort, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. In an illustrative embodiment, an FKHL7 polypeptide is producedrecombinantly utilizing an expression vector generated by sub-cloningthe coding sequence of one of the FKHL7 genes represented in SEQ ID NOS.1 or 3.

The preferred mammalian expression vectors contain both prokaryoticsequences, to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papillomavirus(BPV-1), or Epstein-Barr virus (pHFBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Thevarious methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular Cloning ALaboratory Manual, 2^(nd) Ed., ed. by Sambrook, Fritsch and Maniatis(Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.

In some instances, it may be desirable to express the recombinant FKHL7polypeptide by the use of a baculovirus expression system. Examples ofsuch baculovirus expression systems include pVL-derived vectors (such aspVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1),and pBlueBac-derived vectors (such as the β-gal containing pBlueBac III)

When it is desirable to express only a portion of an FKHL7 protein, suchas a form lacking a portion of the N-terminus, i.e. a truncation mutantwhich lacks the signal peptide, it may be necessary to add a start codon(ATG) to the oligonucleotide fragment containing the desired sequence tobe expressed. It is well known in the art that a methionine at theN-terminal position can be enzymatically cleaved by the use of theenzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli(Ben-Bassat et al (1987) J. Bacteriol 169 751-757) and Salmonellatyphimurium and its in vitro) activity has been demonstrated oilrecombinant proteins (Miller et al. (I 987) PNAS 84:2718-1722).Therefore, removal of an N-terminal methionine, if desired, can beachieved either in vivo by expressing FKHL7 derived polypeptides in ahost which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or invitro by use of purified MAP (e.g., procedure of Miller et al., supra).

Moreover, the gene constructs of the present invention can also be usedas part of a gene therapy protocol to deliver nucleic acids encodingeither an agonistic or antagonistic form of one of the subject FKHL7proteins. Thus, another aspect of the invention features expressionvectors for in vivo or in vitro transfection and expression of an FKHL7polypeptide in particular cell types so as to reconstitute the functionof, or alternatively, abrogate the function of FKHL7 in a tissue. Thiscould be desirable, for example, when the naturally-occurring form ofthe protein is misexpressed or the natural protein is mutated and lessactive.

In addition to viral transfer methods, non-viral methods can also beemployed to cause expression of a subject FKHL7 polypeptide in thetissue of an animal. Most nonviral methods of gene transfer rely onnormal mechanisms used by mammalian cells for the uptake andintracellular transport of macromolecules. In preferred embodiments,non-viral targeting means of the present invention rely on endocyticpathways for the uptake of the subject FKHL7 polypeptide gene by thetargeted cell. Exemplary targeting means of this type include liposomalderived systems, poly-lysine conjugates, and artificial viral envelopes.

In other embodiments, transgenic animals, described in more detail belowcould be used to produce recombinant proteins.

4.4. Polypeptides of the Present Invention

The present invention makes available FKHL7 polypeptides which areisolated from, or otherwise substantially free of other cellularproteins. The term “substantially free of other cellular proteins” (alsoreferred to herein as “contaminating proteins”) or “substantially pureor purified preparations” are defined as encompassing preparations ofFKHL7 polypeptides having less than about 20% (by dry weight)contaminating protein, and preferably having less than about 5%contaminating protein. Functional forms of the subject polypeptides canbe prepared, for the first time, as purified preparations by using acloned gene as described herein.

Preferred FKHL7 proteins of the invention have an amino acid sequencewhich is at least about 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 90%, or 95% identical orhomologous to an amino acid sequence of SEQ ID NO 2. Even more preferredFKHL7 proteins comprise an amino acid sequence which is at least about97, 98, or 99% homologous or identical to an amino acid sequence of SEQID NO. 2. Such proteins can be recombinant proteins, and can be, e.g.,produced in vitro from nucleic acids comprising a nucleotide sequenceset forth in SEQ ID NOS. 1 or 3 or homologs thereof. For example,recombinant polypeptides preferred by the present invention can beencoded by a nucleic acid, which is at least about 85% homologous andmore preferably at least about 90% homologous and most preferably atleast about 95% homologous with a nucleotide sequence set forth in SEQID NOS. 1 or 3. Polypeptides which are encoded by a nucleic acid that isat least about 98-99% homologous with the sequence of SEQ ID NOS. 1 or 3are also within the scope of the invention

In a preferred embodiment, an FKHL7 protein of the present invention isa mammalian FKHL7 protein. In a particularly preferred embodiment anFKHL7 protein is set forth as SEQ ID NO. 2. In particularly preferredembodiments, an FKHL7 protein has an FKHL7 bioactivity. It will beunderstood that certain post-translational modifications, e.g.,phosphorylation and the like, can increase the apparent molecular weightof the FKHL7 protein relative to the unmodified polypeptide chain.

The invention also features protein isoforms encoded by splice variantsof the present invention. Such isoforms may have biological activitiesidentical to or different from those possessed by the FKHL7 proteinsspecified by SEQ ID NO. 2.

FKHL7 polypeptides preferably are capable of functioning as either anagonist or antagonist of at least one biological activity of a wild-type(“authentic”) FKHL7 protein of the appended sequence listing. The term“evolutionarily related to”, with respect to amino acid sequences ofFKHL7 proteins, refers to both polypeptides having amino acid sequencesWhich have arisen naturally, and also to mutational variants of humanFKHL7 polypeptides which are derived, for example, by combinatorialmutagenesis.

Full length proteins or fragments corresponding to one or moreparticular motifs and/or domains or to arbitrary sizes, for example, atleast 5, 10, 25, 50, 75 and 100, amino acids in length are within thescope of the present invention.

For example, isolated FKHL7 polypeptides can be encoded by all or aportion of a nucleic acid sequence salon in any of SEQ ID AMOS. 1 or 3.Isolated peptidyl portions of FKHL7 proteins can be obtained byscreening, peptides recombinantly produced from the correspondingfragment of the nucleic acid encoding such peptides. In addition,fragments can be chemically synthesized using techniques known in theart Such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, an FKHL7 polypeptide of the present inventionmay be arbitrarily divided into fragments of desired length with nooverlap of the fragments, or preferably divided into overlappingfragments of a desired length. The fragments can be produced(recombinantly or by chemical synthesis) and tested to identify thosepeptidyl fragments which can function as either agonists or antagonistsof a wild-type (e.g., “authentic”) FKHL7 protein.

Preferred FKHL7 polypeptides contain the forkhead domain located fromabout amino acid 73 to about 178 of SEQ ID NO. 2 (i.e. the underlinedregion of the protein shown in FIG. 1). Other preferred FKHL7polypeptides bind to an RTAAYA target region of a nucleic acid

In general, polypeptides referred to herein as having an FKHL7 activity(e.g., are “bioactive”) are defined as polypeptides which include anamino acid sequence encoded by all or a portion of the nucleic acidsequences shown in one of SEQ ID NOS. 1 or 3 and which mimic orantagonize all or a portion of the biological/biochemical activities ofa naturally occurring FKHL7 protein. Examples of such biologicalactivity include: regulation of gene expression. Furthermore thesefragments can either promote or inhibit these processes or agonize orantagonize the activity of another agent which itself promotes orinhibits these processes. Other biological activities of the subjectFKHL7 proteins will be reasonably apparent to one of skill in the art.According to the present invention, a polypeptide has biologicalactivity if it is a specific agonist or antagonist of anaturally-occurring form of an FKHL7 protein. Assays for determiningwhether a compound, e g, a protein, such as an FKHL7 protein or variantthereof, has one or more of the above biological activities are wellknown in the art.

Other preferred proteins of the invention are those encoded by thenucleic acids set forth in the section pertaining to nucleic acids ofthe invention. In particular, the invention provides fusion proteins,e.g., FKHL7-immunoglobulin fusion proteins. Such fusion proteins canprovide, e.g., enhanced stability and solubility of FKHL7 proteins andmay thus be useful in therapy. Fusion proteins can also be used toproduce an immunogenic fragment of an FKHL7 protein For example, the VP6capsid protein of rotavirus can be used as an immunologic carrierprotein for portions of the FKHL7 polypeptide, either in the monomericform or in the form of a viral particle. The nucleic acid sequencescorresponding, to the portion of a subject FKHL7 protein to which acidsequences are to be raised can be incorporated into a fusion geneconstruct which includes coding sequences for a late vaccinia virusstructural protein to produce a set of recombinant viruses expressing,fusion proteins comprising FKHL7 epitopes as part of the virion. It hasbeen demonstrated with the use of immunogenic fusion proteins utilizingthe Hepatitis B surface antigen fusion proteins that recombinantHepatitis B virions can be utilized in this role as well. Similarly,chimeric constructs coding for fusion proteins containing a portion ofan FKHL7 protein and the poliovirus capsid protein can be created toenhance immunogenicity of the set of polypeptide antigens (see, forexample, EP Publication No: 0259149; and Evans et al. (1989) Nature339:385; Huang et al. (1988) J. Virol. 62:3855; and Schlienger et al.(1992) J. Virol. 66:2).

The Multiple antigen peptide system for peptide-based immunization canalso be utilized to generate an immunogen, wherein a desired portion ofan FKHL7 polypeptide is obtained directly from organo-chemical synthesisof the peptide onto an oligomeric branching lysine core (see, forexample, Posnett et al. (1988) JBC 263:1719 and Nardelli et al. (1992)J. Immunol. 148:914). Antigenic determinants of FKHL7 proteins can alsobe expressed and presented by bacterial cells.

In addition to itilizing fusion proteins to enhance immunogenicity, itis widely appreciated that fusion proteins can also facilitate theexpression of proteins, and accordingly, can be used in the expressionof the FKHL7 polypeptides of the present invention. For example, FKHL7polypeptides can be generated as glutathione-S-transferase (GST-fusion)proteins Such GST-fusion proteins can enable easy purification of theFKHL7 polypeptide, as for example by the use of glutathione-derivatizedmatrices (see, for example, Current Protocols in Molecular Biology, eds.Ausubel et al. (N.Y.: John Wiley & Sons, 1991)).

The present invention further pertains to methods of producing thesubject FKHL7 polypeptides. For example, a host cell transfected with anucleic acid vector directing expression of a nucleotide sequenceencoding the subject polypeptides can be cultured under appropriateconditions to allow expression of the peptide to occur. Suitable mediafor cell culture are well known in the art. The recombinant FKHL7polypeptide can be isolated from cell culture medium, host cells, orboth using techniques known in the art for purifying proteins includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for such peptides. In a preferred embodiment, therecombinant FKHL7 polypeptide is a fusion protein containing a domainwhich facilitates its purification, such as GST fusion protein.

Moreover, it still be generally appreciate that, under certaincircumstances, it may be advantageous to provide homologs of one of thesubject FKHL7 polypeptides, which function in a limited capacity as oneof either an FKHL7 agonist (mimetic) or an FKHL7 antagonist, in order topromote or inhibit only a subset of the biological activities of thenaturally-occurring form of the protein. Thus, specific biologicaleffects can be elicited by treatment with a homolog of limited function,and with fewer side effects relative to treatment with agonists orantagonists which are directed to all of the biological activities ofnaturally occurring forms of FKHL7 proteins.

Homologs of each of the subject FKHL7 proteins can be generated bymutagenesis, such as by discrete point mutation(s), or by truncation.For instance, mutation can give rise to homologs which retainsubstantially the same, or merely a subset, of the biological activityof the FKHL7 polypeptide from which it was derived. Alternatively,antagonistic forms of the protein can be generated which are able toinhibit the function of the naturally occurring form of the protein,such as by competitively binding to an FKHL7 receptor.

The recombinant FKHL7 polypeptides of the present invention also includehomologs of the wildtype FKHL7 proteins, such as versions of thoseprotein which are resistant to proteolytic cleavage, as for example, dueto mutations which alter ubiquitination or other enzymatic targetingassociated with the protein.

FKHL7 polypeptides may also be chemically modified to create FKHL7derivatives by forming covalent or aggregate conjugates with otherchemical moieties, such as glycosyl groups, lipids, phosphate, acetylgroups and the like. Covalent derivatives of FKHL7 proteins can beprepared by linking the chemical moieties to functional groups on aminoacid sidechains of the protein or at the N-terminus or at the C-terminusof the polypeptide.

Modification of the structure of the subject FKHL7 polypeptides can befor such purposes as enhancing therapeutic or prophylactic efficacy,stability (e g., ex vivo shelf life and resistance to proteolyticdegradation), or post-translational modifications (e g., to alterphosphorylation pattern of protein). Such modified peptides, whendesigned to retain at least one activity of the naturally-occurring formof the protein, or to produce specific antagonists thereof, areconsidered functional equivalents of the FKHL7 polypeptides described inmore detail herein. Such modified peptides can be produced, forinstance, by amino acid substitution, deletion, or addition Thesubstitutional variant may be a substituted conserved amino acid or asubstituted non-conserved amino acid.

For example, it is reasonable to expect that an isolated replacement ofa leucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of amino acid with astructurally related amino acid (i.e. isosteric and/or isoelectricmutations) will not have a major effect on the biological activity ofthe resulting molecule. Conservative replacements are those that takeplace within a family of amino acids that are related in their sidechains. Genetically encoded amino acids can be divided into fourfamilies: (I) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine, (3) nonpolar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. In similarfashion, the amino acid repertoire can be grouped as (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3)aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,threonine, with serine and threonine optionally be grouped separately asaliphatic-hydroxyl, (4) aromatic=phenylalanine, tyrosine, tryptophan;(5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine andmethionine. (see, for example, Biochemistry, 2^(nd) ed., Ed. by L.Stryer, W H Freeman and Co.: 1981). Whether a change in the amino acidsequence of a peptide results in a functional FKHL7 homolog (e g.,functional in the sense that the resulting polypeptide mimics orantagonizes the wild-type form) can be readily determined by assessingthe ability of the variant peptide to produce a response in cells in afashion similar to the wild-type protein, or competitively inhibit sucha response. Polypeptides in which more than one replacement has takenplace can readily be tested in the same manner.

This invention further contemplates a method for generating sets ofcombinatorial mutants of the subject FKHL7 proteins as well astruncation mutants, and is especially useful for identifying potentialvariant sequences (e.g., homologs). The purpose of screening suchcombinatorial libraries is to generate, for example, novel FKHL7homologs which can act as either agonists or antagonist, oralternatively, possess novel activities all together. Thuscombinatorially-derived homologs can be generated to have an increasedpotency relative to a naturally occurring form of the protein.

In one embodiment, the variegated library of FKHL7 variants is generatedby combinatorial mutagenesis at the nucleic acid level, and is encodedby a variegated gene library. For instance, a mixture of syntheticoligonucleotides can be enzymatically ligated into gene sequences suchthat the degenerate set of potential FKHL7 sequences are expressible asindividual polypeptides, or alternatively, as a set of larger fusionproteins (e.g. for phage display) containing the set of FKHL7 sequencestherein

There are many ways by which such libraries of potential FKHL7 homologscan be generated from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequences can be carried out in anautomatic DNA synthesizer, and the synthetic genes then ligated into anappropriate expression vector. The purpose of a degenerate set of genesis to provide, in one mixture, all of the sequences encoding the desiredset of potential FKHL7 sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, SA(1983) Tetrahedron 39:3, Itakura et al. (1981) Recombinant DNA, Proc3^(rd) Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam:Elsevier pp 273-289, Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477. Such techniques have been employed in the directedevolution of other proteins (see, for example, Scott et al. (1990)Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433, Devlin etal. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

Likewise, a library of coding sequence fragments can be provided for anFKHL7 clone in order to generate a variegated population of FKHL7fragments for screening and subsequent selection of bioactive fragments.A variety of techniques are known in the art for generating suchlibraries, including chemical synthesis. In one embodiment, a library ofcoding sequence fragments can be generated by (i) treating a doublestranded PCR fragment of an FKHL7 coding sequence with a nuclease underconditions wherein nicking occurs only about once per molecule, (ii)denaturing the double stranded DNA, (iii) renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products, (iv) removing single stranded portions fromreformed duplexes by treatment with S1 nuclease; and (v) ligating theresulting fragment library into an expression vector. By this exemplarymethod, an expression library can be derived which codes for N-terminal,C-terminal and internal fragments of various sizes

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having acertain property. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of FKHL7 homologs. The most widely used techniques forscreening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. Each of the illustrative assaysdescribed below are amenable to high through-put analysis as necessaryto screen large numbers of degenerate FKHL7 sequences created bycombinatorial mutagenesis techniques. Combinatorial mutagenesis has apotential to generate very large libraries of mutant proteins, e.g., inthe order of 10²⁶ molecules. Combinatorial libraries of this size may betechnically challenging to screen even with high throughput screeningassays. To overcome this problem, a new technique has been developedrecently, recrusive ensemble mutagenesis (REM), which allows one toavoid the very high proportion of non-functional proteins in a randomlibrary and simply enhances the frequency of functional proteins, thusdecreasing the complexity required to achieve a useful sampling ofsequence space. REM is an algorithm which enhances the frequency offunctional mutants in a library when an appropriate selection orscreening method is employed (Arkin and Yourvan, 1992, PNAS USA89:7811-7815, Yourvan et al., 1992, Parallel Problem Solving fromNature, 2., In Maenner and Manderick, eds., Elsevir Publishing Co.,Amsterdam, pp. 401-410; Delgrave et al., 1993, Protein Engineering6(3):327-33 1).

The invention also provides for reduction of the FKHL7 proteins togenerate mimetics, e.g., peptide or non-peptide agents, such as smallmolecules, which are able to disrupt binding of an FKHL7 polypeptide ofthe present invention with a molecule, e.g. target peptide. Thus, suchmutagenic techniques as described above are also useful to map thedeterminants of the FKHL7 proteins which participate in protein-proteininteractions involved in, for example, binding of the subject FKHL7polypeptide to a target peptide. To illustrate, the critical residues ofa subject FKHL7 polypeptide which are involved in molecular recognitionof its receptor can be determined and used to generate FKHL7 derivedpeptidomimetics or small molecules which competitively inhibit bindingof the authentic FKHL7 protein with that moiety. By employing, forexample, scanning mutagenesis to map the amino acid residues of thesubject FKHL7 proteins which are involved in binding other proteins,peptidomimetic compounds can be generated which mimic those residues ofthe FKHL7 protein which facilitate the interaction. Such mimetics ma)then be used to interfere with the normal function of an FKHL7 protein.For instance, non-hydrolyzable peptide analogs of such residues can begenerated using benzodiazepine (e.g., see Freidinger et al. in Peptides:Chemistry and Biology, G R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), azepine (e.g, A See Huffman et al. in peptidesChemistry aid Biology, G. R. Marshall ed., ESCOM Publisher Leiden,Netherlands, 1988), substituted gamma lactam rings (Garvey et al inPeptides Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, (1988), keto-methylene pseudopeptides (Ewenson etal. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structureand Function (Proceedings of the 9^(th) American Peptide Symposium)Pierce Chemical Co. Rockland, Ill., 1985), b-turn dipeptide cores (Nagaiet al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) 3 Chem SocPerkin Trans 1: 1231), and β-aminoalcohols (Gordon et al. (1985) BiochemBiophys Res Commun 126:419; and Dann et al. (1986) Biochem Biophys ResCommun 134:71).

4.5. Anti-FKHL7 Antibodies and Uses Therefor

Another aspect of the invention pertains to an antibody specificallyreactive with a mammalian FKHL7 protein, e.g., a wild-type or mutatedFKHL7 protein. For example, by using immunogens derived from an FKHL7protein, e.g., based on the cDNA sequences, anti-protein/anti-peptideantisera or monoclonal antibodies can be made by standard protocols(See, for example, Antibodies: A Laboratory Manual ed. by Harlow andLane (Cold Spring Harbor Press 1988)). A mammal, such as a mouse, ahamster or rabbit can be immunized with an immunogenic form of thepeptide (e.g., a mammalian FKHL7 polypeptide or an antigenic fragmentwhich is capable of eliciting an antibody response, or a fusion proteinas described above). Techniques for conferring immunogenicity on aprotein or peptide include conjugation to carriers or other techniqueswell known in the art. An immunogenic portion of an FKHL7 protein can beadministered in the presence of adjuvant. The progress of immunizationcan be monitored by detection of antibody titers in plasma or serum.Standard ELISA or other immunoassays can be used with the immunogen asantigen to assess the levels of antibodies. In a preferred embodiment,the subject antibodies are immunospecific for antigenic determinants ofan FKHL7 protein of a mammal, e.g., antigenic determinants of a proteinset forth in SEQ ID No: 2 or closely related homologs (e.g., at least90% homologous, and more preferably at least 941,homologous).

Following immunization of an animal with an antigenic preparation of anFKHL7 polypeptide, anti-FKHL7 antisera can be obtained and, if desired,polyclonal anti-FKHL7 antibodies isolated from the serum. To producemonoclonal antibodies, antibody-producing cells lymphocytes) can beharvested from an immunized animal and fused by standard somatic cellfusion procedures with immortalizing cells such as mycloma cell, toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique originally developed byKohler and Milstein (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985). Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a mammalian FKHL7polypeptide of the present invention and monoclonal antibodies isolatedfrom a culture comprising such hybridoma cells. In one embodimentanti-human FKHL7 antibodies specifically react with the protein encodedby a nucleic acid having SEQ ID NO. 1 or 3.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with one of the subjectmammalian FKHL7 polypeptides. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described above for whole antibodies. For example, F(ab)₂fragments can be generated by treating antibody with pepsin. Theresulting F(ab)₂ fragment can be treated to reduce disulfide bridges toproduce Fab fragments. The antibody of the present invention is furtherintended to include bispecific, single-chain, and chimeric and humanizedmolecules having affinity for an FKHL7 protein conferred by at least oneCDR region of the antibody. In preferred embodiments, the antibodyfurther comprises a label attached thereto and able to be detected,(e.g., the label can be a radioisotope, fluorescent compound, enzyme orenzyme co-factor).

Anti-FKHL7 antibodies can be used, e.g., to monitor FKHL7 protein levelsin an individual for determining. e.g., whether a subject has a diseaseor condition associated with an aberrant FKHL7 protein level, orallowing determination of the efficacy of a given treatment regimen foran individual afflicted with such a disorder, The level of FKHL7polypeptides may be measured from cells in bodily fluid, such as inblood samples.

Another application of anti-FKHL7 antibodies of the present invention isin the immunological screening of cDNA libraries constructed inexpression vectors such as λgt11, λgt18-23, λZAP, and λRF8. Messengerlibraries of this type, having coding sequences inserted in the correctreading frame and orientation, can produce fusion proteins. Forinstance, λgt11 will produce fusion proteins whose amino termini consistof β-galactose amino acid sequences and whose carboxy termini consist ofa foreign polypeptide. Antigenic epitopes of an FKHL7 protein e.g.,other orthologs of a particular FKHL7 protein or other paralogs from thesame species, can then be detected with antibodies, as for example,reacting nitrocellulose fibers lifted from infected plates withanti-FKHL7 antibodies. Positive phage detected by this assay can then beisolated from the infected plate. Thus, the presence of FKHL7 homologscan be detected and cloned from other animals, as can alternate isoforms(including splice variants) from humans.

4.6. Transgenic Animals

The invention further provides for transgenic animals, which can be usedfor a variety of purposes, e.g., to identify FKHL7 therapeutics.Transgenic animals of the invention include non-human animals containinga heterologous FKHL7 gene or fragment thereof under the control of anFKHL7 promoter or under the control of a heterologous promoter.Accordingly, the transgenic animals of the invention can be animalsexpressing a transgene encoding a wild-type FKHL7 protein or fragmentthereof or variants thereof, including mutants and polymorphic variantsthereof. Such animals can be used, e.g., to determine the effect of adifference in amino acid sequence of an FKHL7 protein from the sequenceset forth in SEQ ID NO. 2, such as a polymorphic difference. Theseanimals can also be used to determine the effect of expression of anFKHL7 protein in a specific site or for identifying FKHL7 therapeuticsor confirming their activity ill vivo.

The transgenic animals can also be animals containing a transgene, suchas reporter gene, under the control of an FKHL7 promoter or fragmentthereof. These animals are useful, e.g., for identifying compound thatmodulate production of FKHL7, such as by modulating FKHL7 geneexpression. An FKHL7 gene promoter can be isolated, e.g., by screeningof a genomic library With an FKHL7 cDNA fragment and characterizedaccording to methods known in the art. In a preferred embodiment of thepresent invention, the transgenic animal containing said FKHL7 reportergene is used to screen a class of bioactive molecules known as steroidhormones for their ability to modulate FKHL7 expression. In a morepreferred embodiment of the invention, the steroid hormones screened forFKHL7 expression modulating activity belong to the group known asandrogens In a still more preferred embodiment of the invention, thesteroid hormone is testosterone or a testosterone analog. Yet othernon-human animals within the scope of the invention include those intwhich the expression of the endogenous FKHL7 gene has Seen mutated or“knocked Out”. “knock out” animal is one carrying, a homozygous orheterozygous deletion of a particular gene or genes. These animals couldbe useful to determine whether the absence of FKHL7 will result in aspecific phenotype, in particular whether these mice have or are likelyto develop a specific disease, such as high susceptibility to heartdisease or cancer. Furthermore these animals are useful in screens fordrugs which alleviate or attenuate the disease condition resulting fromthe mutation of the FKHL7 gene as outlined below. These animals are alsouseful for determining the effect of a specific amino acid difference,or allelic variation, in an FKHL7 gene. That is, the FKHL7 knock outanimals can be crossed with transgenic animals expressing, e.g., amutated form or allelic variant of FKHL7, thus resulting in an animalwhich expresses only the mutated protein and not the wild-type FKHL7protein.

In a preferred embodiment of this aspect of the invention, a transgenicFKHL7 knock-out mouse, carrying the mutated FKHL7 locus on one or bothof its chromosomes, is used as a model system for transgenic or drugtreatment of the condition resulting from loss of FKHL7 expression.

Methods for obtaining transgenic and knockout non-human animals are wellknown in the art. Knock out mice are generated by homologous integrationof a “knock out” construct into a mouse embryonic stem cell chromosomewhich encodes the gene to be knocked out. In one embodiment, genetargeting, which is a method of using homologous recombination to modifyan animal's genome, can be used to introduce changes into culturedembryonic stem cells. By targeting a FKHL7 gene of interest in ES cells,these changes can be introduced into the germlines of animals togenerate chimeras. The gene targeting procedure is accomplished byintroducing into tissue culture cells a DNA targeting construct thatincludes a segment homologous to a target FKHL7 locus, and which alsoincludes an intended sequence modification to the FKHL7 genomic sequence(e.g., insertion, deletion, point mutation). The treated cells are thenscreened for accurate targeting to identify and isolate those which havebeen properly targeted.

Gene targeting in embryonic stem cells is in fact a scheme contemplatedby the present invention as a means for disrupting a FKHL7 gene functionthrough the use of a targeting transgene construct designed to undergohomologous recombination with one or more FKHL7 genomic sequences Thetargeting construct can be arranged so that, upon recombination with anelement of a FKHL7 gene, a positive selection marker is inserted into(or replaces) coding sequences of the gene. The inserted sequencefunctionally disrupts the FKHL7 gene, while also providing a positiveselection trait. Exemplary FKHL7 targeted constructs are described inmore detail below.

Generally, the embryonic stem cells (ES cells) used to produce theknockout animals will be of the same species as the knockout animal tobe generated. Thus for examples mouse embryonic stem cells will usuallybe used for generation of knockout mice.

Embryonic stem cells are generated and maintained using methods wellknown to the skilled artisan such as those described by Doetschman etal. (1985) J Embryol. Exp. MoFKHL7hol. 87:27-45). Any line of ES cellscan be used, however, the line chosen is typically selected for theability of the cells to integrate into and become part of the germ lineof a developing embryo so as to create germ line transmission of theknockout construct. Thus, any ES cell line that is believed to have thiscapability is suitable for use herein. One mouse strain that istypically used for production of ES cells, is the 129J strain. AnotherES cell line is murine cell line D3 (American Type Culture Collection,catalog no. CKL 1934) Still another preferred ES cell line is the WW6cell line (loffe et al. (1995) PNAS 92:7357-7361). The cells arecultured and prepared for knockout construct insertion using methodswell known to the skilled artisan, such as those set forth by Robertsonin: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. IRL Press, Washington, D.C. [1987]), by Bradley et al.(1986) Current Topics in Devel. Biol. 20:357-371); and by Hogan et al.(Manipulating the Mouse Embryo. A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (19861).

A knock out construct refers to a uniquely configured fragment ofnucleic acid which is introduced into a stem cell line and allowed torecombine with the genome at the chromosomal locus of the gene ofinterest to be mutated. Thus a given knock out construct is specific fora given gene to be targeted for disruption. Nonetheless, many commonelements exist among these constructs and these elements are well knownin the art. A typical knock out construct contains nucleic acidfragments of not less than about 0.5 kb nor more than about 10.0 kb fromboth the 5′ and the 3′ ends of the genomic locus which encodes the geneto be mutated. These two fragments are separated by an interveningfragment of nucleic acid which encodes a positive selectable marker,such as the neomycin resistance gene (neo^(R)). The resulting nucleicacid fragment, consisting of a nucleic acid from the extreme 5′ end ofthe genomic locus linked to a nucleic acid encoding a positiveselectable marker which is in turn linked to a nucleic acid from theextreme 3′ end of the genomic locus of interest, omits most of thecoding sequence for FKHL7 or other gene of interest, to be knocked out.Wen the resulting construct recombines homologously with the chromosomeat this locus, it results in the loss of the emitted coding sequence,otherwise known as the structural gene, from the genomic locus. A stemcell which such a rare homologous recombination event has taken placecan be selected tier by virtue of the stable integration into the genomeof the nucleic acid of the gene encoding the positive selectable, markerand subsequent selection for cells expressing this marker gene in thepresence of an appropriate drug (neomycin in this example).

Variations on this basic technique also exist and are well known in theart. For example, a “knock-in” construct refers to the same basicarrangement of a nucleic acid encoding a 5′ genomic locus fragmentlinked to nucleic acid encoding a positive selectable marker which inturn is linked to a nucleic acid encoding a 3′ genomic locus fragment,but which differs in that none of the coding sequence is omitted andthus the 5′ and the 3′ genomic fragments used were initially contiguousbefore being disrupted by the introduction of the nucleic acid encodingthe positive selectable marker gene. This “knock-in”type of construct isthus very useful for the construction of mutant transgenic animals whenonly a limited region of the genomic locus of the gene to be mutated,such as a single exon, is available for cloning and geneticmanipulation. Alternatively, the “knock-in” construct can be used tospecifically eliminate a single functional domain of the targetted gene,resulting in a transgenic animal which expresses a polypeptide of thetargetted gene which is defective in one function, while retaining thefunction of other domains of the encoded polypeptide. This type of“knock-in” mutant frequently has the characteristic of a so-called“dominant negative” mutant because, especially in the case of proteinswhich homomultimerize, it can specifically block the action of (or“poison”) the polypeptide product of the wild-type gene from which itwas derived. In a variation of the knock-in technique, a marker gene isintegrated at the genomic locus of interest such that expression of themarker gene comes under the control of the transcriptional regulatoryelements of the targeted gene. A marker gene is one that encodes anenzyme whose activity can be detected (e.g., P-galactosidase), theenzyme substrate can be added to the cells under suitable conditions,and the enzymatic activity can be analyzed. One skilled in the art willbe familiar with other useful markers and the means for detecting theirpresence in a given cell. All such markers are contemplated as beingincluded within the scope of the teaching of this invention.

As mentioned above, the homologous recombination of the above described“knock out” and “knock in” constructs is very rare and frequently such aconstruct inserts nonhomologously into a random region of the genomewhere it has no effect on the gene which has been targeted for deletion,and where it can potentially recombine so as to disrupt another genewhich was otherwise not intended to be altered Such nonhomologousrecombination events can be selected against by modifying theabovementioned knock out and knock in constructs so that they areflanked by negative selectable markers at either end (particularlythrough the use of two allelic variants of the thymidine kinase gene,the polypeptide product of which can be selected against in expressingcell lines in an appropriate tissue culture medium well known in theart—i.e. one containing a drug such as 5-bromodeoxyuridine). Thus apreferred embodiment of such a knock out or knock in construct of theinvention consist of a nucleic acid encoding a negative selectablemarker linked to a nucleic acid encoding a 5′ end of a genomic locuslinked to a nucleic acid of a positive selectable marker which in turnis linked to a nucleic acid encoding a 3′ end of the same genomic locuswhich in turn is linked to a second nucleic acid encoding a negativeselectable marker Nonhomologous recombination between the resultingknock out construct and the genome will usually result in the stableintegration of one or both of these negative selectable marker genes andhence cells which have undergone nonhomologous recombination can beselected against by growth in the appropriate selective media (e.g mediacontaining a drug such as 5-bromodeoxyuridine for example). Simultaneousselection for the positive selectable marker and against the negativeselectable marker will result in a vast enrichment for clones in whichthe knock out construct has recombined homologously at the locus of thegene intended to be mutated. The presence of the predicted chromosomalalteration at the targeted gene locus in the resulting knock out stemcell line can be confirmed by means of Southern blot analyticaltechniques which are well known to those familiar in the art.Alternatively, PCR can be used.

Each knockout construct to be inserted into the cell must first be inthe linear form. Therefore, if the knockout construct has been insertedinto a vector (described infra), linearization is accomplished bydigesting the DNA with a suitable restriction endonuclease selected tocut only within the vector sequence and not within the knockoutconstrict sequence

For insertion, the knockout construct is added to the ES cells underappropriate conditions for the insertion method chosen, as is known tothe skilled artisan.

For example, if the FS cells are to be electroporated, the ES cells andknockout construct DNA are exposed to an electric pulse using anelectroporation machine and following the manufacturer's guidelines foruse. After electroporation, the ES cells are typically allowed torecover tinder suitable incubation conditions The cells are thenscreened for the presence of the knock out construct as explained above.Where more than one construct is to be introduced into the ES cell, eachknockout construct can be introduced simultaneously or one at a time.

After suitable ES cells containing the knockout construct in the properlocation have been identified by the selection techniques describedabove, the cells can be inserted into an embryo. Insertion may beaccomplished in a variety of ways known to the skilled artisan, howevera preferred method is by microinjection. For microinjection, about 10-30cells are collected into a micropipet and injected into embryos that areat the proper stage of development to permit integration of the foreignES cell containing the knockout construct into the developing embryo.For instance, the transformed ES cells can be microinjected intoblastocytes. The suitable stage of development for the embryo used forinsertion of ES cells is very species dependent, however for mice it isabout 3.5 days. The embryos are obtained by perfusing the uterus ofpregnant females. Suitable methods for accomplishing this are known tothe skilled artisan, and are set forth by, e.g., Bradley et al. (supra).

While any embryo of the right stage of development is suitable for use,preferred embryos are male. In mice, the preferred embryos also havegenes coding for a coat color that is different from the coat colorencoded by the ES cell genes. In this way, the offspring can be screenedeasily for the presence of the knockout construct by looking for mosaiccoat color (indicating that the ES cell was incorporated into thedeveloping embryo) Thus, for example, it the ES cell line carries thegenes for white fur, the embryo selected will carry genes for black orbrown fur.

After the ES cell has been introduced into the embryo, the embryo may beimplanted into the uterus of a pseudopregnant foster mother forgestation. While any foster mother may be used, the foster mother istypically selected for her ability to breed and reproduce well, and forher ability to care for the young. Such foster mothers are typicallyprepared by mating with vasectomized males of the same species. Thestage of the pseudopregnant foster mother is important for successfulimplantation, and it is species dependent. For mice, this stage is about2-3 days pseudopregnant.

Offspring that are born to the foster mother may be screened initiallyfor mosaic coat color where the coat color selection strategy (asdescribed above, and in the appended examples) has been employed. Inaddition, or as an alternative, DNA from tail tissue of the offspringmay be screened for the presence of the knockout construct usingSouthern blots and/or PCR as described above. Offspring that appear tobe mosaics may then be crossed to each other, if they are believed tocarry the knockout construct in their germ line, in order to generatehomozygous knockout animals. Homozygotes may be identified by southernblotting of equivalent amounts of genomic DNA from mice that are theproduct of this cross as well as mice that are known heterozygotes andwild type mice.

Other means of identifying and characterizing the knockout offspring areavailable. For example, Northern blots can be used to probe the mRNA forthe presence or absence of transcripts encoding either the gene knockedout, the marker gene, or both. In addition, Western blots can be used toassess the level of expression of the FKHL7 gene knocked out in varioustissues of the offspring by probing the Western blot with an antibodyagainst the particular FKHL7 protein, or an antibody against the markergene product, where this gene is expressed. Finally, in situ analysis(such as fixing the cells and labeling with antibody) and/or FACS(fluorescence activated cell sorting) analysis of various cells from theoffspring can be conducted using suitable antibodies to look for thepresence or absence of the knockout construct gene product.

Yet other methods of making knock-out or disruption transgenic animalsare also generally known. See, for example, Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Recombinase dependent knockouts can also be generated, e.g. byhomologous recombination to insert target sequences, such that tissuespecific and/or temporal control of inactivation of an FKHL7-gene can becontrolled by recombinase sequences (described infra).

Animals containing more than one knockout construct and/or more than onetransgene expression construct are prepared in any of several ways. Thepreferred manner of preparation is to generate a series of mammals, eachcontaining one of the desired transgenic phenotypes Such animals arebred together through a series of crosses, backcrosses and selections,to ultimately generate a single animal containing all desired knockoutconstructs and/or expression constructs, where the animal is otherwisecongenic (genetically identical) to the wild type except for thepresence of the knockout construct(s) and/or transgene(s).

A FKHL7 transgene can encode the wild-type form of the protein, or canencode homologs thereof, including both agonists and antagonists, aswell as anti-sense constructs. In preferred embodiments, the expressionof the transgene is restricted to specific subsets of cells, tissues ordevelopmental stages utilizing, for example, cis-acting sequences thatcontrol expression in the desired pattern. In the present invention,such mosaic expression of a FKHL7 protein can be essential for manyforms of lineage analysis and can additionally provide a means to assessthe effects of, for example, lack of FKHL7 expression which mightgrossly alter development in small patches of tissue within an otherwisenormal embryo. Toward this and, tissue-specific regulatory sequences andconditional regulatory sequences can be used to control epression of thetransgene in certain spatial patterns. Moreover, temporal patternsexpression can be provided by, for example, conditional recombinationsystems or prokaryotic transcriptional regulatory sequences.

Genetic techniques, which allow for the expression of transgenes can beregulated via site-specific genetic manipulation in vivo, are known tothose skilled in the art. For instance, genetic systems are availablewhich allow for the regulated expression of a recombinase that catalyzesthe genetic recombination of a target sequence. As used herein, thephrase “target sequence” refers to a nucleotide sequence that isgenetically recombined by a recombinase. The target sequence is flankedby recombinase recognition sequences and is generally either excised orinverted in cells expressing recombinase activity. Recombinase catalyzedrecombination events can be designed such that recombination of thetarget sequence results in either the activation or repression ofexpression of one of the subject FKHL7 proteins. For example, excisionof a target sequence which interferes with the expression of arecombinant FKHL7 gene, such as one which encodes an antagonistichomolog or an antisense transcript, can be designed to activateexpression of that gene. This interference with expression of theprotein can result from a variety of mechanisms, such as spatialseparation of the FKHL7 gene from the promoter element or an internalstop codon. Moreover, the transgene can be made wherein the codingsequence of the gene is flanked by recombinase recognition sequences andis initially transfected into cells in a 3′ to 5′ orientation withrespect to the promoter element. In such an instance, inversion of thetarget sequence will reorient the subject gene by placing the 5′ end ofthe coding sequence in an orientation with respect to the promoterelement which allow for promoter driven transcriptional activation.

The transgenic animals of the present invention all include within aplurality of their cells a transgene of the present invention, whichtransgene alters the phenotype of the “host cell” with respect toregulation of cell growth, death and/or differentiation Since it ispossible to produce transgenic organisms of the invention utilizing oneor more of the transgene constricts described herein, a generaldescription will be given of the production of transgenic organisms byreferring generally to exogenous genetic material. This generaldescription can be adapted by those skilled in the art in order toincorporate specific transgene sequences into organisms utilizing themethods and materials described below.

In an illustrative embodiment, either the creiloxP recombinase system ofbacteriophage P1 (Lakso et al (1992) PNAS 89, 6232-6236. Orban et al.(1992) PNAS 89:6861-6865) or the FLP recombinase system of saccharomycescerevisiae (O'Gorman et a (1991) Science 251: 1351-1355 publication WO92/15694) can be used to generate in vivo site-specific geneticrecombination systems. Cre recombinase catalyzes the site-specificrecombination of an intervening target sequence located between loxPsequences. loxP sequences are 34 base pair nucleotide repeat sequencesto which the Cre recombinase binds and are required for Cre recombinasemediated genetic recombination. The orientation of loxP sequencesdetermines whether the intervening target sequence is excised orinverted when Cre recombinase is present (Abremski et al. (1984) J.Biol. Chem. 259:1509-1514), catalyzing the excision of the targetsequence when the loxP sequences are oriented as direct repeats andcatalyzes inversion of the target sequence when loxP sequences areoriented as inverted repeats.

Accordingly, genetic recombination of the target sequence is dependenton expression of the Cre recombinase. Expression of the recombinase canbe regulated by promoter elements which are subject to regulatorycontrol, e.g., tissue-specific, developmental stage-specific, inducibleor repressible by externally added agents. This regulated control willresult in genetic recombination of the target sequence only in cellswhere recombinase expression is mediated by the promoter element. Thus,the activation expression of a recombinant FKHL7 protein can beregulated via control of recombinase expression.

Use of the crelloxP recombinase system to regulate expression of arecombinant FKHL7 protein requires the construction of a transgenicanimal containing transgenes encoding both the Cre recombinase and thesubject protein. Animals containing both the Cre recombinase and arecombinant FKHL7 gene can be provided through the construction of“double” transgenic animals. A convenient method for providing suchanimals is to mate two transgenic animals each containing a transgene,e.g., an FKHL7 gene and recombinase gene.

One advantage derived from initially constructing transgenic animalscontaining a FKHL7 transgene in a recombinase-mediated expressibleformat derives from the likelihood that the subject protein whetheragonistic or antagonistic, can be deleterious upon expression in thetransgenic animal In such an instance, a founder population, in whichthe subject transgenic is silent iii all tissues, can be propagated andmaintained. Individuals of this founder population can be crossed withanimals expressing the recombinase in for example, one or more tissuesand/or a desired temporal pattern. Thus, the creation of a founderpopulation in which, for example, an antagonistic FKHL7 transgene issilent will allow the study of progeny from that founder in whichdisruption of FKHL7 mediated induction in a particular tissue or atcertain developmental stages would result in, for example, a lethalphenotype.

Similar conditional transgenes can be provided using prokaryoticpromoter sequences which require prokaryotic proteins to be simultaneousexpressed in order to facilitate expression of the FKHL7 transgene.Exemplary promoters and the corresponding trans-activating prokaryoticproteins are given in U.S. Pat. No. 4,833,080.

Moreover, expression of the conditional transgenes can be induced bygene therapy-like methods wherein a gene encoding the trans-activatingprotein, e.g. a recombinase or a prokaryotic protein, is delivered tothe tissue and caused to be expressed, such as in a cell-type specificmanner. By this method, a FKHL7 transgene could remain silent intoadulthood until “turned on” by the introduction of the trans-activator.

In an exemplar), embodiment, the “transgenic non-human animals” of theinvention are produced by introducing transgenes into the germline ofthe non-human animal. Embryonal target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonal target cell. Thespecific line(s) of any animal used to practice this invention areselected for general good health, good embryo yields, good pronuclearvisibility in the embryo, and good reproductive fitness. In addition,the haplotype is a significant factor. For example, when transgenic miceare to be produced, strains such as C57BL/6 or FVB lines are often used(Jackson Laboratory, Bar Harbor, Me.). Preferred strains are those withH-2^(b), H-2^(d) or H-2^(q) haplotypes such as C57BL/6 or DBA/l. Theline(s) used to practice this invention may themselves be transgenics,and/or may be knockouts (i.e., obtained from animals which have one ormore genes partially or completely suppressed).

In one embodiment, the transgene construct is introduced into a singlestage embryo. The zygote is the best target for micro-injection. In themouse, the male pronucleus reaches the size of approximately 20micrometers in diameter which allows reproducible injection of 1-2 pl ofDNA solution The use of zygotes as a target for gene transfer has amajor advantage in that in most cases the injected DNA will beincorporated into the host gene before the first cleavage (Brinster etal. (1985) PNAS 82:4438-4442). As a consequence, all cells of thetransgenic animal will carry the incorporated transgenic. This will ingeneral also be reflected in the efficient transmission of the transgeneto offsprings of the founder since 50% of the cells will harbor thetransgene.

Normally, fertilized embryos are incubated in suitable media until thepronuclei appear At about this time, the nucleotide sequence comprisingthe transgene is introduced into the female or male pronucleus asdescribed below. In some species such as mice, the male pronucleus ispreferred. It is most preferred that the exogenous genetic material beadded to the male DNA complement of the zygote prior to its beingprocessed by the ovum nucleus or the zygote female pronucleus. It isthought that the ovum nucleus or female pronucleus release moleculeswhich affect the male DNA complement, perhaps by replacing theprotamines of the male DNA with histones, thereby facilitating thecombination of the female and male DNA complements to form the diploidzygote

Thus, it is preferred that the exogenous genetic material be added tothe male complement of DNA or any other complement of DNA prior to itsbeing affected by the female pronucleus For example, the exogenousgenetic material is added to the early male pronucleus, as soon aspossible after the formation of the male pronucleus, which is when themale and female pronuclei are well separated and both are located closeto the cell membrane. Alternatively, the exogenous genetic materialcould be added to the nucleus of the sperm after it has been induced toundergo decondensation. Sperm containing the exogenous genetic materialcan then be added to the ovum or the decondensed sperm could be added tothe ovum with the transgene constructs being added as soon as possiblethereafter.

Introduction of the transgene nucleotide sequence into the embryo may beaccomplished by any means known in the art such as, for example,microinjection, electroporation, or lipofection. Following introductionof the transgene nucleotide sequence into the embryo, the embryo may beincubated in vitro for varying amounts of time, or reimplanted into thesurrogate host, or both. In vitro incubation to maturity is within thescope of this invention. One common method in to incubate the embryos invitro for about 1-7 days, depending on the species, and then reimplantthem into the surrogate host.

For the purposes of this invention a zygote is essentially the formationof a diploid cell which is capable of developing into a completeorganism. Generally, the zygote will be comprised of an egg containing anucleus formed, either naturally or artificially, by the fusion of twohaploid nuclei from a gamete or gametes. Thus, the gamete nuclei must beones which are naturally compatible, i.e., ones which result in a viablezygote capable of undergoing differentiation and developing into afunctioning organism. Generally, a euploid zygote is preferred. If ananeuploid zygote is obtained, then the number of chromosomes should notvary by more than one with respect to the eaploid number of the organismfrom which either gamete originated

In addition to similar biological considerations, physical ones alsogovern the amount (e.g., volume) of exogenous genetic material which canbe added to the nucleus of the zygote or to the genetic material whichforms a part of the zygote nucleus. If no genetic material is removed,then the amount of exogenous genetic material which can be added islimited by the amount which will be absorbed without being physicallydisruptive. Generally, the volume of exogenous genetic material insertedwill not exceed about 10 picoliters. The physical effects of additionmust not be so great as to physically destroy the viability of thezygote. The biological limit of the number and variety of DNA sequenceswill vary depending upon the particular zygote and functions of theexogenous genetic material and will be readily apparent to one skilledin the art, because the genetic material, including the exogenousgenetic material, of the resulting zygote must be biologically capableof initiating and maintaining the differentiation and development of thezygote into a functional organism.

The number of copies of the transgene constructs which are added to thezygote is dependent upon the total amount of exogenous genetic materialadded and will be the amount which enables the genetic transformation tooccur. Theoretically only one copy is required; however, generally,numerous copies are utilized, for example, 1,000-20,000 copies of thetransgene construct, in order to insure that one copy is functional. Asregards the present invention, there will often be an advantage tohaving more than one functioning copy of each of the inserted exogenousDNA sequences to enhance the phenotypic expression of the exogenous DNAsequences.

Any technique which allows for the addition of the exogenous geneticmaterial into nucleic genetic material can be utilized so long as it isnot destructive to the cell, nuclear membrane or other existing cellularor genetic structures. The exogenous genetic material is preferentiallyinserted into the nucleic genetic material by microinjection.Microinjection of cells and cellular structures is known and is used inthe art.

Reimplantation is accomplished using standard methods. Usually, thesurrogate host is anesthetized, and the embryos are inserted into theoviduct. The number of embryos implanted into a particular host willvary by species, but will usually be comparable to the number of offspring the species naturally produces.

Transgenic offspring the surrogate host may be screened for the presenceand/or expression of the transgene by any suitable method. Screening isoften accomplished by Southern blot or Northern blot analysis using aprobe that is complementary to it lest a portion of the transgene.Western blot analysis using an antibody against the protein encoded bythe transgene may be employed as an alternative or additional method forscreening for the presence of the transgene product. Typically, DNA isprepared from tail tissue and analyzed by Southern analysis or PCR forthe transgene. Alternatively, the tissues or cells believed to expressthe transgene at the highest levels are tested for the presence andexpression of the transgene using Southern analysis or PCR, although anytissues or cell types may be used for this analysis.

Alternative or additional methods for evaluating the presence of thetransgene include, without limitation, suitable biochemical assays suchas enzyme and/or immunological assays, histological stains forparticular marker or enzyme activities, flow cytometric analysis, andthe like. Analysis of the blood may also be useful to detect thepresence of the transgene product in the blood, as well as to evaluatethe effect of the transgene on the levels of various types of bloodcells and other blood constituents.

Progeny of the transgenic animals may be obtained by mating thetransgenic animal with a suitable partner, or by iii vitro fertilizationof eggs and/or sperm obtained from the transgenic animal. Where mating %with a partner is to be performed, the partner may or may not betransgenic and/or a knockout, where it is transgenic, it may contain thesame or a different transgene, or both Alternatively, the partner may bea parental line. Where in vitro fertilization is used, the fertilizedembryo may be implanted into a surrogate host or incubated in vitro, orboth. Using either method, the progeny may be evaluated for the presenceof the transgene using methods described above, or other appropriatemethods.

The transgenic animals produced in accordance with the present inventionwill include exogenous genetic material. As set out above, the exogenousgenetic material will, in certain embodiments, be a DNA sequence whichresults in the production of a FKHL7 protein (either agonistic orantagonistic), and antisense transcript, or a FKHL7 mutant. Further, insuch embodiments the sequence will be attached to a transcriptionalcontrol element. e.g. a promoter, which preferably allows the expressionof the transgene product in a specific type of cell.

Retroviral infection can also be used to introduce transgene into anon-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage During this time, the blastomeres can betargets for retroviral infection (Jacnich R (1976) PNAS 73 1260-1264)Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Manipulating the Mouse Embryo,Hogan eds (Cold Spring Harbor laboratory Press. Cold Spring Harbor,1986) The viral vector system used to introduce the transgene istypically a replication-defective retrovirus carrying the transgene(Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985)PNAS 82′:6148-6152). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal. (1982) Nature 298:623-628). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infection of the midgestation embryo(Jahner et al (1982) supra).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (Evans et al. (1981) Nature292:154-156; Bradley et al. (1984) Nature 309:255-258, Gossler et al.(1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature322:445-448). Transgenes can be efficiently introduced into the ES cellsby DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocytes from anon-human animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. For reviewsee Jaenisch, R. (1988) Science 240: 1468-1474.

4.7. Screening Assays for FKHL7 Therapeutics

The invention further provides screening methods for identifying FKHL7therapeutics, e.g. for treating and/or preventing the development of acongenital heart disease.

An FKHL7 therapeutic can be any type of compound, including a protein, apeptide, peptidomimetic, small molecule, and nucleic acid. A nucleicacid can be, e.g., an FKHL7 gene, an antisense nucleic acid, a ribozyme,or a triplex molecule. An FKHL7 therapeutic of the invention can be anagonist or an antagonist. Preferred FKHL7 agonists include FKHL7 genesor proteins or derivatives thereof which mimic at least one FKHL7activity. Others preferred agonists include compounds which ire capableof increasing the production of an FKHL7 protein in a cell, e.g.,compounds capable of upregulating the expression of an FKHL7 gene, andcompounds which are capable of enhancing an FKHL7 activity and/or theinteraction of an FKHL7 protein with another molecule, such as a targetpeptide. Preferred FKHL7 antagonists include FKHL7 proteins which aredominant negative proteins. Other preferred antagonists includecompounds which decrease or inhibit the production of an FKHL7 proteinin a cell and compounds which are capable of downregulating expressionof an FKHL7 gene, and compounds which are capable of downregulating anFKHL7 activity and/or interaction of an FKHL7 protein with anothermolecule. In another preferred embodiment, an FKHL7 antagonist is amodified form of a target peptide, which is capable of binding to agene, but which does not regulate expression of the gene.

The invention also provides screening methods for identifying FKHL7agonist and antagonist compounds, comprising selecting compounds whichare capable of interacting with an FKHL7 protein or with a moleculecapable of interacting with an FKHL7 protein. In general, a moleculewhich is capable of interacting with an FKHL7 protein is referred toherein as “FKHL7 binding partner”.

The compounds of the invention can be identified using various assaysdepending on the type of compound and activity of the compound that isdesired. In addition, as described herein, the test compounds can befurther tested in animal models. Set forth below are at least someassays that can be used for identifying FKHL7 therapeutics. However,based on the instant disclosure, one of skill in the art could useadditional assays for identifying FKHL7 therapeutics without requiringundue experimentation.

4.7.1 Cell-Free Assays

Cell-free assays can be used to identify compounds which are capable ofinteracting with an FKHL7 protein or binding partner, to thereby modify,the activity of the FKHL7 protein or binding partner. Such a compoundcan, e g., modify the structure of an FKHL7 protein or binding partnerand thereby effect its activity. Cell-free assays can also be used toidentify compounds which modulate the interaction between an FKHL7protein and an FKHL7 binding partner, such as a target peptide. In apreferred embodiment, cell-free assays for identifying such compoundsconsist essentially in a reaction mixture containing an FKHL7 proteinand a test compound or a library of test compounds in the presence orabsence of a binding partner. A test compound can be, e.g., a derivativeof an FKHL7 binding partner, e.g., a biologically inactive targetpeptide, or a small molecule.

Accordingly, one exemplary screening assay of the present inventionincludes the steps of contacting an FKHL7 protein or functional fragmentthereof or an FKHL7 binding partner with a test compound or library oftest compounds and detecting the formation of complexes. For detectionpurposes, the molecule can be labeled with a specific marker and thetest compound or library of test compounds labeled with a differentmarker. Interaction of a test compound with an FKHL7 protein or fragmentthereof or FKHL7 binding partner can then be detected by determining thelevel of the two labels after an incubation step and a washing step. Thepresence of two labels after the washing step is indicative of aninteraction.

An interaction between molecules can also be identified by usingreal-time BIA (Biomolecular Interaction Analysis, Pharmacia BiosensorAB) which detects surface plasmon resonance (SPR), an opticalphenomenon. Detection depends on changes in the mass concentration ofmacromolecules at the biospecific interface, and does not require anylabeling of interactants In one embodiment, a library of test compoundscan be immobilized on a sensor surface, e.g., which forms one wall of amicro-flow cell. A solution containing the FKHL7 protein, functionalfragment thereof, FKHL7 analog or FKHL7 binding partner is then flowncontinuously over the sensor surface A change in the resonance angle asshown on a signal recording, indicates that an interaction has occurred.This technique is further described, e.g., in BIA technology Handbook byPharmacia.

Another exemplary screening assay of the present invention includes thesteps of (a) forming a reaction mixture including (i) an FKHL7polypeptide, (ii) an FKHL7 binding partner, and (iii) a test compound;and (b) detecting interaction of the FKHL7 and the FKHL7 bindingprotein. The FKHL7 polypeptide and FKHL7 binding partner can be producedrecombinantly, purified from a source, e.g., plasma, or chemicallysynthesized, as described herein. A statistically significant change(potentiation or inhibition) in the interaction of the FKHL7 and FKHL7binding protein in the presence of the test compound, relative to theinteraction in the absence of the test compound, indicates a potentialagonist (mimetic or potentiator) or antagonist (inhibitor) of FKHL7bioactivity for the test compound The compounds of this assay can becontacted simultaneously. Alternatively. an FKHL7 protein can first becontacted with a test compound for an appropriate amount of time,following which the FKHL7 binding partner is added to the reactionmixture. The efficacy of the compound can be assessed by generating doseresponse curves from data obtained using various concentrations of thetest compound. Moreover, a control assay can also be performed toprovide a baseline for comparison. In the control assay, isolated andpurified FKHL7 polypeptide or binding partner is added to a compositioncontaining the FKHL7 binding partner or FKHL7 polypeptide, and theformation of a complex is quantitated in the absence of the testcompound.

Complex formation between an FKHL7 protein and an FKHL7 binding partnermay be detected by a variety of techniques. Modulation of the formationof complexes can be quantitated using, for example, detectably labeledproteins such as radiolabeled, fluorescently labeled, or enzymaticallylabeled FKHL7 proteins or FKHL7 binding partners, by immunoassay, or bychromatographic detection.

Typically, it will be desirable to immobilize either FKHL7 or itsbinding partner to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay. Binding of FKHL7 to an FKHL7 binding partner,can be accomplished in any vessel suitable for containing the reactants.Examples include microtitre plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows the protein to be bound to a matrix. For example,glutathione-S-transferase/FKHL7 (GST/FKHL7) fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the FKHL7 binding partner, e.g. an ³⁵S-labeled FKHL7binding partner, and the test compound, and the mixture incubated underconditions conducive to complex formation, e.g. at physiologicalconditions for salt and FKHL7 though slightly more stringent conditionsmats be desired. Following incubation, the beads are washed to removeany unbound label, and the matrix immobilized and radiolabel determineddirectly (e.g. beads placed in scintilant), or in the supernatant afterthe complexes are subsequently dissociated. Alternatively, the complexescan be dissociated from the matrix, separated by SDS-PAGE, and the levelof FKHL7 protein or FKHL7 binding partner found in the bead fractionquantitated from the gel using standard electrophoretic techniques suchas described in the appended examples.

Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assays. For instance either FKHL7 orits cognate binding partner can be immobilized utilizing conjugation ofbiotin and streptavidin. For instance, biotinylated FKHL7 molecules canbe prepared from biotin-NHS (N-hydroxy-succinimide) using techniqueswell known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical). Alternatively, antibodies reactive withcan be derivatized to the wells of the plate, and FKHL7 trapped in thewells by antibody conjugation. As above, preparations of an FKHL7binding protein and a test compound are incubated in the FKHL7presenting wells of the plate, and the amount of complex trapped in thewell can be quantitated. Exemplary methods for detecting such complexes,in addition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with theFKHL7 binding partner, or which are reactive with FKHL7 protein andcompete with the binding partner, as well as enzyme-linked assays whichrely on detecting an enzymatic activity associated with the bindingpartner, either intrinsic or extrinsic activity. In the instance of thelatter, the enzyme can be chemically conjugated or provided as a fusionprotein with the FKHL7 binding partner. To illustrate, the FKHL7 bindingpartner can be chemically cross-linked or genetically fused withhorseradish peroxidase, and the amount of polypeptide trapped in thecomplex can be assessed with a chromogenic substrate of the enzyme, e.g.3,3′-diamino-benzidine tetrahydrochloride or 4-chloro-1-napthol.Likewise, a fusion protein comprising the polypeptide andglutathione-S-transferase can be provided, and complex formationquantitated by detecting the GST activity using1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 2499:7130).

For processes which rely on immunodetection for quantitating one of theproteins trapped in the complex, antibodies against the protein, such asanti-FKHL7 antibodies, can be used. Alternatively, the protein to bedetected in the complex can be “epitope tagged” in the form of a fusionprotein which includes, in addition to the FKHL7 sequence, a secondpolypeptide for which antibodies are readily available (e.g. fromcommercial sources) For instance, the GST fusion proteins describedabove can also be used for quantification of binding using antibodiesagainst the GST moiety. Other useful epitope tags include myc-epitopes(e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) whichincludes a residue sequence from c-myc, as well as the pFLAG system(International Biotechnologies, Inc.) or the pEZZ-protein A system(Pharmacia, N.J.).

Cell-free assays can also be used to identify, compounds which interactwith an FKHL7 protein and modulate an activity of an FKHL7 protein.Accordingly, in one embodiment an FKHL7 protein is contacted wits a testcompound and the catalytic activity of FKHL7 is monitored. In oneembodiment, the ability of FKHL7 to bind a target molecule isdetermined. The binding affinity of FKHL7 to a target molecule can bedetermined according to methods known in the art. Determination of theenzymatic activity of FKHL7 can be performed with the aid of thesubstrate furanacryloyl-L-phenylalanyl-glycyl-glycine (FAPGG) underconditions described in Holmquist et al. (1979) Anal. Biochem. 95:540and in U.S. Pat. No. 5,259,045.

4.7.2. Cell Based Assays

In addition to cell-free assays, such as described above, FKHL7 proteinsas provided by the present invention, facilitate the generation ofcell-based assays, e.g., for identifying small molecule agonists orantagonists. Cell based assays can be used, for example, to identifycompounds which modulate expression of an FKHL7 gene, modulatetranslation of an FKHL7 mRNA, which modulate the stability of an FKHL7mRNA or protein or which otherwise interfere with an interaction betweenan FKHL7 gene or protein and an FKHL7 binding partner. Accordingly, inone embodiment, a cell which is capable of producing FKHL7 is incubatedwith a test compound and the amount of FKHL7 produced in the cell mediumis measured and compared to that produced from a cell which has not beencontacted with the test compound. The specificity of the compound vis avis FKHL7 can be confirmed by various control analysis, e.g., measuringthe expression of one or more control genes. Compounds which can betested include small molecules, proteins, and nucleic acids. Inparticular, this assay can be used to determine the efficacy of FKHL7antisense molecules or ribozymes.

In another embodiment, the effect of a test compound on transcription ofan FKHL7 gene is determined by transfection experiments using a reportergene operatively linked to at least a portion of the promoter of anFKHL7 gene. A promoter region of a gene can be isolated, e.g., from agenomic library according to methods known in the art. The reporter genecan be any gene encoding a protein which is readily quantifiable, e.g,the luciferase or CAT gene. Such reporter gene are well known in theart.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

4.8 Predictive Medicine

The invention further features predictive medicines, which are based, atleast in part. on the identity of the novel FKHL7 gene and alterationsin the genes and related pathway genes, which affect the expressionlevel and/or function of the encoded FKHL7 protein in a subject.

For example, as described herein, FKHL7 mutations that are particularlylikely to cause or contribute to the development of a congenital heartdisease are those mutations that negatively impact normal (wildtype)functioning of the forkhead domain that is involved with the DNA bindingproperties of FKHL7. Examples of such mutations include: i) upstreammutations that encode truncated transcripts that lack the DNA-binding,forkhead domain (e.g. an 11 base pair deletion encoding an FKHL7transcript that is missing 477 amino acids), and ii) missense mutationsoccurring within the forkhead domain (e.g a cytosine to thyminetransition that causes an amino acid change at position 131 from serineto leucine (Ser 131Leu); a cytosine to guanine transition that causes anamino acid change at position 126 from isoleucine to methionine(Ile126Met); and a thymine to cytosine transition, which results in areplacement of phenylalanine with serine at position 112 (Phe112Ser) Inaddition, mutations or translocations that result in expression of onlyone cops of FHKL7 (e.g. monosomy of 6p25), can result in a congenitalheart disease phenotype.

Information obtained using the diagnostic assays described herein (aloneor in conjunction with information on another genetic defect, whichcontributes to the same disease) is useful for prognosing, diagnosing orconfirming that a subject has a genetic defect (e.g. in an FKHL7 gene orin a gene that regulates the expression of an FKHL7 gene), which causesor contributes to the development of glaucoma Based on prognosticinformation, a doctor can recommend a regimen (e.g. diet or exercise) ortherapeutic protocol, Which is useful for preventing or prolonging onsetof congenital heart disease in the individual.

In addition, knowledge of the particular alteration or alterations,resulting in defective or deficient FKHL7 genes or proteins in anindividual (the FKHL7 genetic profile), alone or in conjunction withinformation on other genetic defects contributing to a congenital heartdisease (the congenital heart disease genetic profile) allowscustomization of therapy to the individual's genetic profile, the goalof “pharmacogenomics”. For example, an individual's FKHL7 geneticprofile or the congenital heart disease genetic profile can enable adoctor to 1) more effectively prescribe a drug ratio that will addressthe molecular basis of the glaucoma, and 2) better determine theappropriate dosage of a particular drug for the particular individual.For example, the expression level of FKHL7 proteins, alone or inconjunction with the expression level of other genes, known tocontribute to the same disease, can be measured in many patients atvarious stages of the disease to generate a transcriptional orexpression profile of the disease. Expression patterns of individualpatients can then be compared to the expression profile of the diseaseto determine the appropriate drug and dose to administer to the patient.

The ability to target populations expected to show the highest clinicalbenefit, based on the FKHL7 or disease genetic profile, can enable: 1)the repositioning of marketed drugs with disappointing market results;2) the rescue of drug candidates whose clinical development has beendiscontinued as a result of safety or efficacy limitations, which arepatient subgroup-specific; and 3) an accelerated and less costlydevelopment for drug candidates and more optimal drug labeling (e.g.since the use of FKHL7 as a marker is, useful for optimizing effectivedose)

These and other methods are described in further detail in the followingsections.

4.8.1. Prognostic and Diagnostic Assays

The present methods provide means for determining if a subject has(diagnostic) or is at risk of developing (prognostic) a disease,condition or disorder that is associated with an aberrant FKHL7activity, e.g., an aberrant level of FKHL7 protein or an aberrantbioactivity, such as results in the development of a congenital heartdisease.

Accordingly, the invention provides methods for determining whether asubject has or is likely to develop a congenital heart disease,comprising determining the level of an FKHL7 gene or protein, an FKHL7bioactivity and/or the presence of a mutation or particular polymorphicvariant in the FKHL7 gene.

In one embodiment, the method comprises determining whether a subjecthas an abnormal mRNA and/or protein level of FKHL7, such as by Northernblot analysis, reverse transcription-polymerase chain reaction (RT-PCR),in situ hybridization, immunoprecipitation, Western blot hybridization,or immunohistochemistry. According to the method, cells are obtainedfrom a subject and the FKHL7 protein or mRNA level is determined andcompared to the level of FKHL7 protein or mRNA level in a healthysubject. An abnormal level of FKHL7 polypeptide or mRNA level is likelyto be indicative of an aberrant FKHL7 activity,

In another embodiment, the method comprises measuring at least oneactivity of FKHL7. For example, regulation of the expression of a geneby an FKHL7 can be determined, e.g., as described herein. Comparison ofthe results obtained with results from similar analysis performed onFKHL7 proteins from healthy subjects is indicative of whether a subjecthas an abnormal FKHL7 activity.

In preferred embodiments, the methods for determining whether a subjecthas or is at risk for developing a disease, which is caused by orcontributed to by an aberrant FKHL7 activity is characterized ascomprising detecting, in a sample of cells from the subject, thepresence or absence of a genetic alteration characterized by at leastone of (i) an alteration affecting the integrity of a gene encoding anFKHL7 polypeptide, or (ii) the mis-expression of the FKHL7 gene. Forexample, such genetic alterations can be detected by ascertaining theexistence of at least one of: (i) a deletion of one or more nucleotidesfrom an FKHL7 gene, (ii) an addition of one or more nucleotides to anFKHL7 gene, (ii) a substitution of one or more nucleotides of an FKHL7gene, (iv) a gross chromosomal rearrangement of an FKHL7 gene, (v) agross alteration in the level of a messenger RNA transcript of an FKHL7gene, (vi) aberrant modification of an FKHL7 gene, such as of themethylation pattern of the genomic DNA, (vii) the presence of a non-wildtype splicing pattern of a messenger RNA transcript of an FKHL7 gene,(viii) a non-wild type level of an FKHL7 polypeptide, (ix) allelic lossof an FKHL7 gene, and/or (x) inappropriate post-translationalmodification of an FKHL7 polypeptide. As set out below, the presentinvention provides a large number of assay techniques for detectingalterations in an FKHL7 gene. These methods include, but are not limitedto, methods involving sequence analysis, Southern blot hybridization,restriction enzyme site mapping, and methods involving ing detection ofthe absence of nucleotide pairing between the nucleic acid to beanalyzed and a probe. These and other methods are further describedinfra.

Specific diseases or disorders, e.g., genetic diseases or disorders, areassociated with specific allelic variants of polymorphic regions ofcertain genes, which do not necessarily encode a mutated proteins. Thus,the presence of a specific allelic variant of a polymorphic region of agene such as a single nucleotide polymorphism (“SNP”), in a subject canrender the subject susceptible to developing a specific disease ordisorder. Polymorphic regions in genes, e.g. FKHL7 genes, can beidentified, by determining the nucleotide sequence of genes inpopulations of individuals. If a polymorphic region, e.g. SNP isidentified, then the link with specific disease can be determined bystudying specific populations of individuals, e.g. individuals whichdeveloped a specific disease, such as glaucoma A polymorphic region canbe located in any region of a gene, e.g., exons, in coding or non codingregions of exons, introns, and promoter region.

It is likely that FKHL7 genes comprise polymorphic regions, specificalleles of which may be associated with specific diseases or conditionsor with an increased likelihood of developing such diseases orconditions. Thus, the invention provides methods for determining theidentity of the allele or allelic variant of a polymorphic region of anFKHL7 gene in a subject, to thereby determine whether the subject has oris at risk of developing a disease or disorder that is associated with aspecific allelic variant of a polymorphic region.

In an exemplary embodiment, there is provided a nucleic acid compositioncomprising a nucleic acid probe including a region of nucleotidesequence which is capable of hybridizing to a sense or antisensesequence of an FKHL7 gene or naturally occurring mutants thereof, or 5′or 3′ flanking sequences naturally associated with the subject FKHL7genes or naturally occurring mutants thereof. The nucleic acid of a cellis rendered accessible for hybridization, the probe is contacted withthe nucleic acid of the sample, and the hybridization of the probe tothe sample nucleic acid is detected. Such techniques can be used todetect alterations or allelic variants at either the genomic or mRNAlevel, including deletions, substitutions, etc., as well as to determinemRNA transcript levels.

A preferred detection method is allele specific hybridization usingprobes overlapping the mutation or polymorphic site and having about 5,10, 20, 25, or 30 nucleotides around the mutation or polymorphic region.In a preferred embodiment of the invention, several probes capable ofhybridizing specifically to allelic variants, such as single nucleotidepolymorphisms, are attached to a solid phase support, e.g., a “chip”.Oligonucleotides can be bound to a solid support by a variety ofprocesses, including lithography. For example a chip can hold up toabout 250,000 oligonucleotides. Mutation detection analysis using thesechips comprising oligonucleotides, also termed “DNA probe arrays” isdescribed e.g., in Cronin et al. (1996) Human Mutation 7:244. In oneembodiment, a chip comprises all the allelic variants of at least onepolymorphic region of a gene. The solid phase support is then contactedwith a test nucleic acid and hybridization to the specific probes isdetected. Accordingly, the identity of numerous allelic variants of oneor more genes can be identified in a simple hybridization experiment.

In certain embodiments, detection of the alteration comprises utilizingthe probe/primer in a polymerase Chain reaction (PCR) (see, e.g. U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligase chain reaction (LCR) (see, e.g., Landegran etal. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS 91360-364), the latter of which can be particularly useful for detectingpoint mutations in the FKHL7 gene (see Abravaya et al. (1995) Nuc AcidRes 23:675-682). In a merely illustrative embodiment, the methodincludes the steps of (i) collecting a sample of cells from a patient,(ii) isolating nucleic acid (e.g., genomic, mRNA or both) from the cellsof the sample, (iii) contacting the nucleic acid sample with one or moreprimers which specifically hybridize to an FKHL7 gene under conditionssuch that hybridization and amplification of the FKHL7 gene (if present)occurs, and (iv) detecting the presence or absence of an amplificationproduct, or detecting the size of the amplification product andcomparing the length to a control sample. It is anticipated that PCR,LCR or an) other amplification procedure (e.g. self sustained sequencereplication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), or Q-Beta Replicase(Lizardi, P. M. et al., 1988, Bio/Technology 6:1197)), may be used as apreliminary step to increase the amount of sample on which can beperformed, any of the techniques for detecting mutations describedherein.

In a preferred embodiment of the subject assay, mutations in, or allelicvariants, of an FKHL7 gene from a sample cell are identified byalterations in restriction enzyme cleavage patterns. For example, sampleand control DNA is isolated, amplified (optionally), digested with oneor more restriction endonucleases, and fragment length sizes aredetermined by gel electrophoresis. Moreover, the use of sequencespecific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the FKHL7 gene anddetect mutations by comparing the sequence of the sample FKHL7 with thecorresponding wild-type (control) sequence. Exemplary sequencingreactions include those based on techniques developed by Maxim andGilbert (Proc. Natl. Acad Sci. USA (1977) 74:560) or Sanger (Sanger etal (1977) Proc. Natl. Acad. Sci. 74:5463). It is also contemplated thatany of a variety of automated sequencing procedures may be utilized whenperforming the subject assays. (Biotechniques (1995) 19 448), includingsequencing by mass spectrometry (see, for example PCT publication WO94/16101, Cohen et al (1996) Adv Chromatogr 36:127-162, and Griffin etal. (1993) Appl. Biochem Biotechnol 38:147-159). It will be evident toone skilled in the art that for certain embodiments, the occurrence ofonly one, two or three of the nucleic acid bases need be determined inthe sequencing reaction. For instance, A-track or the like, e.g., whereonly one nucleic acid is detected, can be carried out.

In a further embodiment, protection from cleavage agents (such as anuclease, hydroxylamine or osmium tetroxide and with piperidine) can beused to detect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNAheteroduplexes (Myers, et al. (1985) Science 230:1242). In general, theart technique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing (labelled) RNA or DNA containing the wild-typeFKHL7 sequence with potentially mutant RNA or DNA obtained from a tissuesample. The double-stranded duplexes are treated with an agent whichcleaves single-stranded regions of the duplex such as which will existdue to base pair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digest the mismatched regions.In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treatedwith hydroxylamine or osmium tetroxide and with piperidine in order todigest mismatched regions. After digestion of the mismatched regions,the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, for example,Cotton et al (1988) Proc. Natl Acad Sci USA 85:4397: Saleeba et al.(1992) Methods Enzymol. 217:286-295. In a preferred embodiment, thecontrol DNA or RNA can be labeled for detection

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in FKHL7 cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15 1657-1662)According to an exemplary embodiment, a probe based on an FKHL7 sequencee.g., a wild-type FKHL7 sequence, is hybridized to a cDNA or other DNAproduct from a test cell(s). The duplex is treated with a DNA mismatchrepair enzyme, and the cleavage products, if any, can be detected fromelectrophoresis protocols or the like See, for example. U.S. Pat. No.5,409,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations or the identity of the allelic variant, of apolymorphic region in FKHL7 genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766, see also Cotton(1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl9:73-79). Single-stranded DNA fragments of sample and control FKHL7nucleic acids are denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may belabelled or detected with labelled probes The sensitivity of the assaymay be enhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 70.5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al (1985)Nature 313:495). When DGGE is used as the method of analysis, DNA willbe modified to insure that it does not completely denature, for exampleby adding a GC clamp of approximately 40 bp of high-melting GC-rich DNAby PCR. In a further embodiment, a temperature gradient is used in placeof a denaturing agent gradient to identify differences in the mobilityof control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem265:12753).

Examples of other techniques for detecting point mutations or theidentity of the allelic variant of a polymorphic region include, but arenot limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation ornucleotide difference (e.g., in allelic variants) is placed centrallyand then hybridized to target DNA under conditions which permithybridization only if a perfect match is found (Saiki et al (1986)Nature 324:163), Saiki et al (1989) Proc. Natl Acad. Sci USA 86:6230).Such allele specific oligonucleotide hybridization techniques may beused to test one mutation or polymorphic region per reaction whenoligonucleotides are hybridized to PCR amplified target DNA or a numberof different mutations or polymorphic regions when the oligonucleotidesare attached to the hybridizing membrane and hybridized with labeledtarget DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjugation with the instantinvention Oligonucleotides used as primers for specific amplificationmay, carry the mutation or polymorphic region of interest in the centerof the molecule (so that amplification depends on differentialhybridization) (Gibbs et al (1989) Nucleic Acids Res. 17:2437-2448) orat the extreme 3′ end of one primer where, under appropriate conditions,mismatch can prevent, or reduce polymerase extension (Prossner (1993)Tibtech 11:238. In addition it may be desirable to introduce a novelrestriction site in the region of the mutation to create cleavage-baseddetection (Gasparini et al (1992) Mol Cell Probes 6: 1). It isanticipated that in certain embodiments amplification may also beperformed using Taq ligase for amplification (Barany (1991) Proc. Natl.Acad. Sci USA 88:189). In such cases, ligation will occur only if thereis a perfect match at the 3′ end of the 5′ sequence making it possibleto detect the presence of a known mutation at a specific site by lookingfor the presence or absence of amplification.

In another embodiment, identification of the allelic variant is carriedout using an oligonucleotide ligation assay (OLA), as described, e.g.,in U.S. Pat. No. 4,998,617 and in Landegren, U. et al., Science 2411077-1080 (1988). The OLA protocol uses two oligonucleotides which aredesigned to be capable of hybridizing to abutting sequences of a singlestrand of a target One of the oligonucleotides is linked to a separationmarker, e.g., biotinylated, and the other is detectably labeled. If theprecise complementary sequence is found in a target molecule, theoligonucleotides will hybridize such that their termini abut, and createa ligation substrate. Ligation then permits the labeled oligonucleotideto be recovered using avidin, or another biotin ligand. Nickerson, D. A.et al. have described a nucleic acid detection assay that combinesattributes of PCR and OLA (Nickerson, D A. et al., Proc. Natl. Acad.Sci. (U.S.A.) 87:8923-8927 (1990). In this method, PCR is used toachieve the exponential amplification of target DNA, which is thendetected using OLA

Several techniques based on this OLA method have been developed and canbe used to detect specific allelic variants of a polymorphic region ofan FKHL7 gene. For example, U.S. Pat. No 5,593,826 discloses an OLAusing an oligonucleotide having 3′-amino group and a 5′-phosphorylatedoligonucleotide to form a conjugate having a phosphoramidate linkage Inanother variation of OLA described in To be et al. ((1996) Nucleic AcidsRes 24 3728). OLA combined with PCR permits typing of two alleles in asingle microtiter well. By marking each of the allele-specific primerswith a unique hapten, i.e. digoxigenin and fluorescein, each OLAreaction can be detected by using hapten specific antibodies that arelabeled with different enzyme reporters, alkaline phosphatase orhorseradish peroxidase. This system permits the detection of the twoalleles using a high throughput format that leads to the production oftwo different colors.

The invention further provides methods for detecting single nucleotidepolymorphisms in an FKHL7 gene. Because single nucleotide polymorphismsconstitute sites of variation flanked by regions of invariant sequence,their analysis requires no more than the determination of the identityof the single nucleotide present at the site of variation and it isunnecessary to determine a complete gene sequence for each patient.Several methods have been developed to facilitate the analysis of suchsingle nucleotide polymorphisms.

In one embodiment, the single base polymorphism can be detected by usinga specialized exonuclease-resistant nucleotide, as disclosed, e.g., inMundy, C. R. (U.S. Pat. No. 4,656,127). According to the method, aprimer complementary to the allelic sequence immediately 3′ to thepolymorphic site is permitted to hybridize to a target molecule obtainedfrom a particular animal or human. If the polymorphic site on the targetmolecule contains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

In another embodiment of the invention, a solution-based method is usedfor determining the identity of the nucleotide of a polymorphic site.Cohen, D. et al. (French Patent 2,650,840; PCT Appln No. WO91/02087). Asin the Mundy method of U.S. Pat. No. 4,656,127, a primer is employedthat is complementary to allelic sequences immediately 3′ to apolymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

An alternative method, known as Genetic Bit Analysis or GBA™ isdescribed by Goelet. P et al (PCT Appln No. 92/15712) The method ofGoelet, P. et al uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3′ to a polymorphic site The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase.

Recently, several primer-guided nucleotide incorporation procedures forassaying polymorphic sites in DNA have been described (Komher, J. S. etal., Nucl. Acids. Res. 17:7779-7784 (1989), Sokolov, B. P., Nucl. AcidsRes. 18:3671 (1990); Syvanen, A. -C., et al., Genomics 8:684-692 (1990);Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88 1143-1147(1991), Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992), Ugozzoli,L. et al., GATA 9:107-112 (1992), Nyren, P. et al., Anal. Biochem.208:171-175 (1993)). These methods differ from GBA™ in that they allrely on the incorporation of labeled deoxynucleotides to discriminatebetween bases at a polymorphic site. In such a format, since the signalis proportional to the number of deoxynucleotides incorporated,polymorphisms that occur in runs of the same nucleotide can result insignals that are proportional to the length of the run (Syvanen, A. -C.,et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

For mutations that produce premature termination of protein translation,the protein truncation test (PTT) offers an efficient diagnosticapproach (Roest, et. al., (1993) Hum. Mol. Genet. 2: 1719-21 van derLuijt, et. al., (1994′) Genomics 20:1-4). For PTT, RNA is initiallyisolated from available tissue and reverse-transcribed, and the segmentof interest is amplified by PCR. The products of reverse transcriptionPCR are then used as a template for nested PCR amplification with aprimer that contains an RNA polymerase promoter and a sequence forinitiating eukaryotic translation. After amplification of the region ofinterest, the unique motifs incorporated into the primer permitsequential in vitro transcription and translation of the PCR products.Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis oftranslation products, the appearance of truncated polypeptides signalsthe presence of a mutation that causes premature termination oftranslation In a variation of this technique, DNA (as opposed to RNA) isused as a PCR template when the target region of interest is derivedfrom a single exon.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acid,primer set; and/or antibody reagent described herein, which may beconventionally used, e.g., in clinical settings to diagnose patientsexhibiting symptoms or family history of a disease or illness involvingFKHL7 polypeptide.

Any cell type or tissue may be utilized in the diagnostics describedbelow. In a preferred embodiment a bodily fluid, e.g., blood, isobtained from the subject to determine the presence of a mutation or theidentity of the allelic variant of a polymorphic region of an FKHL7gene. A bodily fluid, e.g., blood, can be obtained by known techniques(e.g. venipuncture). Alternatively, nucleic acid tests can be performedon dry samples (e.g. hair or skin). For prenatal diagnosis, fetalnucleic acid samples can be obtained from maternal blood as described inInternational Patent Application No. WO91/07660 to Bianchi.Alternatively, amniocytes or chorionic villi may be obtained forperforming prenatal testing.

When using RNA or protein to determine the presence of a mutation or ofa specific allelic variant of a polymorphic region of an FKHL7 gene, thecells or tissues that may be utilized must express the FKHL7 gene.Preferred cells for use in these methods include cardiac cells (seeExamples). Alternative cells or tissues that can be used, can beidentified by determining the expression pattern of the specific FKHL7gene in a subject, such as by Northern blot analysis.

Diagnostic procedures may also be performed in suit directly upon tissuesections (fixed and/or frozen) of patient tissue obtained from biopsiesor resections, such that no nucleic acid purification is necessary,Nucleic acid reagents may be used as probes and/or primers for such insitu procedures (see, for example, Nuovo, G. J., 1992, PCR in sitehybridization: protocols and applications, Raven Press, N.Y.).

In addition to methods which focus primarily on the detection of onenucleic acid sequence, profiles may also be assessed in such detectionschemes. Fingerprint profiles may be generated, for example, byutilizing a differential display procedure, Northern analysis and/orRT-PCR.

Antibodies directed against wild type or mutant FKHL7 polypeptides orallelic variants thereof which are discussed above, may also be used indisease diagnostics and prognostics. Such diagnostic methods, may beused to detect abnormalities in the level of FKHL7 polypeptideexpression, or abnormalities in the structure and/or tissue, cellular,or subcellular location of an FKHL7 polypeptide. Structural differencesmay include, for example, differences in the size, electronegativity, orantigenicity of the mutant FKHL7 polypeptide relative to the normalFKHL7 polypeptide. Protein from the tissue or cell type to be analyzedmay easily be detected or isolated using techniques which are well knownto one of skill in the art, including but not limited to western blotanalysis. For a detailed explanation of methods for carrying out Westernblot analysis, see Sambrook et al. 1989, supra, it Chapter 18. Theprotein detection and isolation methods employed herein may also be suchas those described in Harlow and Lane, for example, (Harlow, E. andLane, D., 1988, “Antibodies: A Laboratory Manual”, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), which is incorporatedherein by reference in its entirety.

This can be accomplished, for example, by immunofluorescence techniquesemploying a fluorescently labeled antibody (see below) coupled withlight microscopic, flow cytometric, or fluorimetric detection. Theantibodies (or fragments thereof) useful in the present invention may,additionally, be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of FKHL7 polypeptides.In situ detection may be accomplished by removing a histologicalspecimen from a patient, and applying thereto a labeled antibody of thepresent invention. The antibody (or fragment) is preferably applied byoverlaying the labeled antibody (or fragment) onto a biological sample.Through the use of such a procedure, it is possible to determine notonly the presence of the FKHL7 polypeptide, but also its distribution inthe examined tissue. Using the present invention, one of ordinary skillwill readily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchill situ detection.

Often a solid phase support or carrier is used as a support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

One means for labeling an anti-FKHL7 polypeptide specific antibody isvia linkage to an enzyme and use in an enzyme immunoassay (EIA) (Voller,“The Enzyme linkage immunosorbent Assay (ELISA)”, Diagnostic Horizons2:1-7, 1978, Microbiological Associates Quarterly Publication,Walkersville, Md., Voller, et al., J. Clin. Pathol. 31 507-520(1978),Butler, Meth. Enzymol. 73:482-523 (1981), Maggio, (ed.) EnzymeImmunoassay, CRC Press, Boca Raton Fl. 1980 Ishikawa, et al., (eds.)Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981). The enzyme which is boundto the antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietywhich can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes which can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect fingerprint gene wild typeor mutant peptides through the use of a radioimmunoassay (RIA) (see, forexample, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,March, 1986, which is incorporated by reference herein). The radioactiveisotope can be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, O-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting, the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

Moreover, it will be understood that any of the above methods fordetecting alterations in a gene or gene product or polymorphic variantscan be used to monitor the course of treatment or therapy.

4.8.2. Pharmacogenomics

Knowledge of the particular alteration or alterations, resulting indefective or deficient FKHL7 genes or proteins in an individual (theFKHL7 genetic profile), alone or in conjunction with information onother genetic defects contributing to the same disease (the geneticprofile of the particular disease) allows a customization of the therapyfor a particular disease to the individual's genetic profile, the goalof “pharmacogenomics”. For example, subjects having a specific allele ofan FKHL7 gene may or may not exhibit symptoms of a particular disease orbe predisposed of developing symptoms of a particular disease. Further,if those subjects are symptomatic, they may or may not respond to acertain drug, e.g., a specific FKHL7 therapeutic, but may respond toanother. Thus, generation of an FKHL7 genetic profile, (e.g.,categorization of alterations in FKHL7 genes which are associated withthe development of glaucoma), from a population of subjects, who aresymptomatic for a disease or condition that is caused by or contributedto by a defective and/or deficient FKHL7 gene and/or protein (an FKHL7genetic population profile) and comparison of an individual's FKHL7profile to the population profile, permits the selection or design ofdrugs that are expected to be safe and efficacious for a particularpatient or patient population (i.e., a group of patients having the samegenetic alteration).

For example, an FKHL7 population profile can be performed, bydetermining, the FKHL7 profile, e.g. the identity of FKHL7 genes, in apatient population having a disease, which is caused by or contributedto by a defective or deficient FKHL7 gene. Optionally, the FKHL7population profile can further include information relating to theresponse of the population to an FKHL7 therapeutic, using any of avariety of methods, including, monitoring: 1) the severity of symptomsassociated with the FKHL7 related disease, 2) FKHL7 gene expressionlevel, 3) FKHL7 mRNA level, and/or 4) FKHL7 protein level, and (iii)dividing or categorizing the population based on the particular geneticalteration or alterations present in its FKHL7 gene or an FKHL7 pathwaygene. The FKHL7 genetic population profile can also, optionally,indicate those particular alterations in which the patient was eitherresponsive or non-responsive to a particular therapeutic. Thisinformation or population profile, is then useful for predicting whichindividuals should respond to particular drugs, based on theirindividual FKHL7 profile.

In a preferred embodiment, the FKHL7 profile is a transcriptional orexpression level profile and step (i) is comprised of determining theexpression level of FKHL7 proteins, alone or in conjunction with theexpression level of other genes, known to contribute to the samedisease. The FKHL7 profile can be measured in many patients at variousstages of the disease.

Pharmacogenomic studies can also be performed using transgenic animals.For example, one can produce transgenic mice, e.g., as described herein,which contain a specific allelic variant of an FKHL7 gene. These micecan be created, e.g, by replacing their wild-type FKHL7 gene with anallele of the human FKHL7 gene. The response of these mice to specificFKHL7 therapeutics can then be determined

4.8.3. Monitoring of Effects of FKHL7 Therapeutics During ClinicalTrials

The ability to target populations expected to show the highest clinicalbenefit, based on the FKHL7 or disease genetic profile, can enable 1)the repositioning of marketed drugs with disappointing market results;2) the rescue of drug candidates whose clinical development has beendiscontinued as a result of safety or efficacy limitations, which arepatient subgroup-specific; and 3) an accelerated and less costlydevelopment for drug candidates and more optimal drug labeling (e.g.since the use of FKHL7 as a marker is useful for optimizing effectivedose).

The treatment oft an individual with an FKHL7 therapeutic can bemonitored by determining FKHL7 characteristics, such as FKHL7 proteinlevel or activity FKHL7 mRNA level, and/or FKHL7 transcriptional level.This measurements will indicate whether the treatment is effective orwhether it should be adjusted or optimized. Thus, FKHL7 can be used as amarker for the efficacy of a drug during clinical trials.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (i) obtaininga preadministration sample from a subject prior to administration of theagent; (ii) detecting the level of expression of an FKHL7 protein, mRNA,or genomic DNA in the preadministration sample, (iii) obtaining one ormore post-administration samples from the subject, (iv) detecting thelevel of expression or activity of the FKHL7 protein, mRNA, or genomicDNA in the post-administration samples; (v) comparing the level ofexpression or activity of the FKHL7 protein, mRNA, or genomic DNA in thepreadministration sample with the FKHL7 protein, mRNA, or genomic DNA inthe post administration sample or samples and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of FKHL7 to higher levels than detected, i.e., toincrease the effectiveness of the agent. Alternatively, decreasedadministration of the agent may be desirable to decrease expression oractivity of FKHL7 to lower levels than detected, i.e., to decrease theeffectiveness of the agent.

Cells of a subject may also be obtained before and after administrationof an FKHL7 therapeutic to detect the level of expression of genes otherthan FKHL7, to verify that the FKHL7 therapeutic does not increase ordecrease the expression of genes which could be deleterious. This can bedone, e.g., by using the method of transcriptional profiling Thus, mRNAfrom cells exposed in vivo to an FKHL7 therapeutic and mRNA from thesame type of cells that were not exposed to the FKHL7 therapeutic couldbe reverse transcribed and hybridized to a chip containing DNA fromnumerous genes, to thereby compare the expression of genes in cellstreated and not treated with an FKHL7-therapeutic. If, for example anFKHL7 therapeutic turns on the expression of a proto-oncogene in anindividual, use of this particular FKHL7 therapeutic may be undesirable.

4.8.4 Kits

The invention further provides kits for use in diagnostics or prognosticmethods for glaucoma or for determining which FKHL7 therapeutic shouldbe administered to a subject, for example, by detecting the presence ofFKHL7 mRNA or protein in a biological sample. For example, the kit cancomprise a labeled compound or agent capable of detecting FKHL7 proteinor mRNA in a biological sample; means for determining the amount ofFKHL7 in the sample; and means for comparing the amount of FKHL7 in thesample with a standard. The compound or agent can be packaged in asuitable container. The kit can farther comprise instructions for usingthe kit to detect FKHL7 mRNA or protein. Such a kit can comprise, e.g.,one or more nucleic acid probes capable of hybridizing specifically toat least a portion of an FKHL7 gene or allelic variant thereof, ormutated form thereof.

4.9. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject having a congenital heart disease.Subjects at risk for such a disease can be identified by a diagnostic orprognostic assay, e.g., as described herein. Administration of aprophylactic agent can occur prior to the manifestation of symptomscharacteristic of the FKHL7 aberrancy, such that development of thecongenital heart disease is prevented or, alternatively, delayed in itsprogression. In general, the prophylactic or therapeutic methodscomprise administering to the subject an effective amount of a compoundwhich is capable of agonizing a wildtype FKHL7 activity or antagonizinga mutant (defective) FKHL7 activity. Examples of suitable compoundsinclude the antagonists, agonists or homologues described in detailherein.

4.9.1. Effective Dose

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining The Ld₅₀ (The Dose Lethal To 50% Of ThePopulation) And The Ed50 (the dose therapeutically effective ill 50%,oof the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic induces are preferred. Whilecompounds that exhibit toxic effects may be used, care should be takento design a delivery system that targets such compounds to the site ofaffected tissue in order to minimize potential damage to uninfectedcells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

4.9.2. Formulation and Use

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the compoundsand their physiologically acceptable salts and solvates may beformulated for administration by, for example, injection, inhalation orinsulation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For such therapy, the compounds of the invention can be formulated for avariety of loads of administration, including systemic and topical orlocalized administration Techniques and formulations generally may befound in Remmington's Pharmaceutical Sciences, Meade Publishing Co.,Easton, Pa. For systemic administration, injection is preferred,including intramuscular, intravenous, intraperitoneal, and subcutaneous.For injection, the compounds of the invention can be formulated inliquid solutions, preferably in physiologically compatible buffers suchas Hank's solution or Ringer's solution. In addition, the compounds maybe formulated in solid form and redissolved or suspended immediatelyprior to use. Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia) non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. For buccal administration thecompositions may take the form of tablets or lozenges formulated inconventional manner. For administration by inhalation, the compounds foruse according to the present invention are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insulator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents Alternatively, the activeingredient may be in powder form for constitution Keith a suitablevehicle, e.g. sterile pyrogen-free water, before use

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Other suitable delivery systems includemicrospheres which offer the possibility of local noninvasive deliveryof drugs over an extended period of time. This technology utilizesmicrospheres of precapillary size which can be injected via a coronarycatheter into any selected part of the e.g. heart or other organswithout causing inflammation or ischemia. The administered therapeuticis slowly released from these microspheres and taken up by surroundingtissue cells (e.g. endothelial cells).

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration bile salts and fusidic acidderivatives in addition, detergents may be used to facilitatepermeation. Transmucosal administration may be through nasal sprays orusing suppositories. For topical administration, the oligomers of theinvention are formulated into ointments, salves, gels, or creams asgenerally known in the art. A wash solution can be used locally to treatan injury or inflammation to accelerate healing.

In clinical settings, a gene delivery system for the therapeutic FKHL7gene can be introduced into a patient by any of a number of methods,each of which is familiar in the art. For instance a pharmaceuticalpreparation of the gene delivery system can be introducedsystematically, e.g., by intravenous injection, and specifictransduction of the protein in the target cells occurs predominantlyfrom specificity of transfection provided by the gene delivery, vehicle,cell-type or tissue-type expression due to the transcriptionalregulatory sequences controlling expression of the receptor gene, or acombination thereof. In other embodiments, initial delivery of therecombinant gene is more limited with introduction into the animal beingquite localized. For example, the gene delivery vehicle can beintroduced by catheter (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (e.g., Chen et al (1994) PNAS 91: 3054-3057). An FKHL7 gene,such as any one of the sequences represented in the group consisting ofSEQ ID NOS. 1 and 3 or a sequence homologous thereto can be delivered ina gene therapy constrict by electroporation using techniques described,for example, by Dev et al. ((1994) Cancer Treat Rev 20:105-115).

The pharmaceutical preparation of the gene therapy construct or compoundof the invention can consist essentially of the gene delivery system inan acceptable diluent, or can comprise a slow release matrix in whichthe gene delivery vehicle or compound is imbedded. Alternatively, wherethe complete gene delivery system can be produced intact fromrecombinant cells, e.g., retroviral vectors, the pharmaceuticalpreparation can comprise one or more cells which produce the genedelivery system.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references (including literature references, issued patents,published patent applications as cited throughout this application arehereby expressly incorporated by reference. The practice of the presentinvention will employ, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. See, for example, Molecular Cloning A Laboratory Manual,2^(nd) Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989), DNA Cloning, volumes I and II (D. N. Glovered., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis etal. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization(B. D. Hames & S.J. Higgins eds. 1984); Transcription And Translation (B D Hames & S. J.Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan RLiss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (19S4), the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M P. Calos eds., 1987,Cold Spring Harbor laboratory); Methods In Enzymology, Vols 154 and 155(Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I--V (D. M. Weir and C. C. Blackwell,eds., 1986); Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

5. EXAMPLES

5.1. Cloning and Analysis of Human FKHL7

Methods

Construction of Somatic Cell Hybrids. Lymphoblastoid cell lines (LCLs)were established whole blood from the two translocations patients.Somatic cell hybrids were created from the LCLs of patient with thebalanced translocation using a modification of previously publishedprotocols (Puck, J. M. et al, J. Clin. Invest. 79: 1395-1400(1987),Nussbaum, R. L. et al., Hum. Genet. 64: 148-150 (1983)). Briefly, LCLswere expanded to roughly 2-5×10⁷ cells in RPMI 1640 media with 10%inactivated fetal calf serum. The were pelleted at 1200 g in a table topcentrifuge and resuspended in 2 m. of Dulbecco's Modified Eagles Mediumwith 10% uninactivated fetal calf serum (DMEM/UFCS). The plate was thenincubated overnight in 4 ml of DMEM/UFCS

The following day, the cells were trypsinized and split 1:5 into 100 mmplates. The cells grown in 5 ml of DMEM/UFC supplemented with 10⁻⁴Mhypoxanthine and 4×10⁻⁵M azaserine. This supplemented media was placedas needed until the colonies started to appear (2 to 4 weekspost-fusion). The individual colonies were allowed to grow until theywere clearly visible without magnification. They were then removed fromthe plate using cloning rings to avoid contamination of the hybrid fromothers on the plate and put in 12-well tissue culture plates.

Marker typing. PCR amplification for the analysis of short tandem repeatpolymorphisms (STRPs) was performed rising 20 ng of genomic DNA in 5-plreactions contain 0.5 μl of 10×PCR buffer [100 mM Tris-HCl (pH 8.8), 500mM MgCl₂ 0.01% gelatin (w/v)], 200 μm each of dATP, dCTP, dGTP and dTTP,2.5 pmol of each primer and 0.2 unit of Taq polymerase (BMB, ISC).Samples were subjected to 35 cycles of 94° C. as required) for 30 s and72° C. for 30s. Amplification products were electrophoresed on 6%polyacrylamide gels contain 7.7 M urea at 60 W for approximately 2 h.The bands are detected by silver staining (Bassam, B. J., et al., Anal.Biochem 196: 80-83 (1991))

Marker taping for physical mapping performed on 2% agarose gels using aPCR reaction Size 10 μl. Reaction conditions were as described abovewith the following exception. For markers which proved difficult toamplify using the standard Taq polymerase, we substituted an equalamount of AmpliTaq (ABI) along with an initial incubation of the PCRmixture at 94° C. for 10 m. For the amplification of the FKHL7fragments, 10% DMSO was also added to the reaction mixture. For PCRreactions involving YAC, BAC or plasmid DNA, 1 to 2 ng of DNA wasutilized as template. For colony PCR, a small number of cells wereinoculated into 20 μl of ddH₂O. 1 μl of this suspension was used astemplate for the PCR reaction.

Oligonucleotide primers for the STRPs were obtained as MapPairs(Research Genetics). The custom primers required for this study weredesigned using the PRIMER 0.5 program and synthesized commercially(Research Genetics). Primer sequences for the screening assay andexpected amplification sizes are available on request. Size standardsfor the 2% agarose gels were 100 bp ladder (Gibco/BRL) and for thedenaturing acrylamide gels a 50 bp ladder (Gibco/BRL). For the 0.8%agarose gels, lambda DNA digested with StyT was used as a size marker.

YAC, BAC and cDNA Identification. Initial YACs were identified bysearching a database at the Whitehead Institute/MIT Genome Center(http://www-genome.wi.mit.edu) (Hudson, T J. el gal, Science 270:1945-1954 (1995) with STSs known to be in the 6p25 and 13q22 regionSubsequently, YACs and BACs were identified by a PCR-based screeningassay of pooled libraries (Research Genetics) using various STSs withineach region A few of the chimeric YACs that were in critical areas werealso obtained from a second source (Genome Systems). cDNA clones wereidentified by a BLASTN search of the public dbEST database availablethrough a web interface (http://www.ncbi.nlm.nih.gov).

DNA Isolation. DNA was prepared from the somatic cell hybrid cell linesusing a rapid salt isolation procedure (Laitinen, J. et al.,Biotechniques 17: 316, 318, 320-322) The initial screening of the celllines utilized a 500 ol volume of cells, while for the second stage ofthe DNA preparation the entire contents of a T75 flask was used. YAC DNAwas isolated using the DNA-Pure yeast genomic kit (CPG Inc.). BAC DNAwas prepared via an alkaline lysis protocol as implemented in the VizardPlus Miniprep Kit (Promega) with the following modification to theprotocol. Instead of loading the supernatant onto a vacuum column, it asprecipitated with a 2×volume of absolute EtOH. In addition, 150 μlvolumes were used for the commercial solutions in place of the 200 μlvolumes suggested in the protocol. The precipitated DNA % a then washedwith 70% EtOH and dried. The DNA pellet was then resuspended in 50 μl ofddH₂O. Finally, plasmid DNA was prepared using a Wizard Plus Miniprepkit (Promega) following the recommended protocol. Culture sizes for DNApreparation from YACs, BACs and plasmids were 1.5 ml of the appropriatemedia and antibiotics for each construct.

Subcloning of BACs. BAC DNA was digested with either EcORI or HindIIIfor 8 h at 37° C. in a 50 μl reaction volume. Vector DNA (pUC19) wasalso digested with either EcORI or HindIII under similar conditions. Allrestriction digests were purified by drop dialysis against ddH₂O usingVS filters with a pore size of 0.025 μM (Millipore) for 15 minutes.Integrity of the digest was verified by get electrophoresis of a portionof the reaction on 0.8% agarose gels. Equal amounts of digested BAC DNAand pUC19 vector were mixed and ligated overnight at 14° C. 1 to 3 μl ofligation mix was transformed into DH5α competent cells (Gibco/BRL).Recombinant clones were selected and the inserts were characterized byrestriction enzyme digestion.

Sequencing plasmids and PCR products. PCR products for sequencing wereamplified in a 50 μl reaction size and purified using the Quiaquick PCRClean-up kit (Promega). 500 ng of plasmid DNA (in 4.5 μl) or 4.5 μl ofpurified PCR product was used as template for a sequencing reaction. 1μl of primer (20 pmoles) and 4.5 μl of terminator sequencing mix(Amersham) was added for a final reaction size of 10 μl. Cyclingconditions were performed as specified by the manufacturer. Thesequencing reactions were precipitated in the presence of linearacrylamide and resuspended in 2 ol of loading buffer. The reactions wereanalyzed on an ABI 377 using a run time of 3 h.

Gene Identification nd Characterization. Raw SCF files from ABI 373A and377 sequences were imported directly into the Sequencher v3.0 program(GeneCodes). Contigs were generated by comparing all fragments in aproject with the parameters of at least a 50 hp overlap in sequence witha 75% level of homology. Genomic sequence of both the 6p25 and 13q22regions were submitted to the BLAST server at NCBI for a BLASTN analysison both the R and dbEST databases. Any region which gave a significantscore (p<10⁻⁵) was also submitted for a BLASTX screen of the SWISS-PROTdatabase. EST sequence was obtained from GENBANK and SCF files from theWashU-Merck ftp site (ftp://genome.wusd.edu).

RNA Isolation and Analysis. Freshly dissected embryos and adult tissuesfrom NIH Swiss mice were rapidly frozen in liquid nitrogen and stored at−70° C. until use Total cellular RNA was prepared using RNA STAT −60(Tel-Test “B”, Inc.) according to the manufacturer's specifications.Poly (A) mRNA was isolated using a Poly (A) Quick mRNA Isolation Kit(Stratagene). Two μg of poly (A) mRNA were electrophoresed through a0.8% agarose gel containing formaldehyde. RNA length standards (0.4-9.5kb) were obtained from Gibco-BRL. The gel was stained with ethidiumbromide, destained overnight in 0.1 M ammonium acetate and the RNA wastransferred to Gene Screen Plus (NEN) following the manufacturer'sspecifications.

Hybridization probes were gel-purified inserts of the followingplasmids: human FKHL7 cDNA corresponding to the 3′ UTR (I.M.A.G.E.Consortium Clone ID 864392, Research Genetics), murine Fkhl7 cDNAcorresponding to the same region (I.M.A.G.E. Consortium Clone ID 864300,Research Genetics), the murine cDNA homologue of mannose deyhdratase(I.M.A.G.E. Consortium Clone ID 717347, Research Genetics) and murineB-actin (Clontech). Hybridization probes were labeled with ³²P-(dCTP)and hybridized for 16 h at 42° C. in 50% formamide, 5×SSC(SCC is astandard saline citrate: 0.15 M NaCl. 0.015 M Na citrate), 1× Denhardt'ssolution, 20 mM phosphate buffer (pH 7.6), 1% sodium dodecyl sulfate(SDS), 100 μg/ml salmon sperm DNA and 10% dextran sulfate. The filterwas then washed twice at room temperature in 1×SSC followed by 2 rinsesat 65° C. in 1×SSC−1% SDS and a final room temperature wash in 0.1×SSC.Kodac XAR-5 film was exposed at −70° C. with Dupont Cronex LightningPlus intensifying screens (Dupont). Following autoradiography, thefilter was stripped of radioactivity and subsequently rehybridized

Mutation Detection, and Confirmation. Mutation detection was performedusing single strand conformation polymorphism (SSCP) analysis and directsequencing of PCR products, PCR products were electrophoresed on SSCPgels (5 ml glycerol, 5 ml 5×TBE, 12.5 ml 37.5:1 acrylamide/bis and 77.5ml ddH₂o) for 3 to 4 hr in 0.25×TBE at room temperature. Gels weresilver stained as described above, Abnormal variants were sequenced andcompared to a control sample to detect any changes from that of thenormal sequence. Mutations were confirmed by amplification-refractorymutation system (ARMS) analysis (Newton, C. R. et al., Nucleic AcidsRes. 17: 2503-2516 (1989).

Results

Clinical features of translocation patients An infant female wasdelivered at 38 weeks gestation with an apparent de novo balancedtranslocation 46,XX,t(6;13)(p25.3;q22.3). She was noted to have a numberof congenital anomalies including a small mandible, cleft palate,hypoplastic lungs segmental abnormalities of the cervical spine, andagenesis of the corpus callosum. Eye findings included nasolacrimal ductobstruction, persistent tumea vasculosa lentis, lower lid epiblepharon,ectropion, fistula to the nasolacrimal system, fat prolapse in the lefteye and hypertension. She was diagnosed with PCG at the age of 6 months.Her parents and siblings are phenotypically normal and her parents havenormal karyotypes.

Cytogenetic evaluation of a second infant female presenting withmultiple congenital anomalies (cardiac defects, poor muscle tone,craniofacial abnormalities and hydronephrosis) revealed an unbalancedtranslocation: 46,XX,der(6)t(2;6)(q35;p25) with the loss of the region6p25−>pter and gain of 2q35−>qter. At 5 days of age, she was found tohave PCG based on diffuse corneal haze, presence of posteriorembryotoxon, increased axial eye lengths, barely visible irides andelevated intraocular pressures.

Since the rearrangements in the above two patients appeared to occur inthe same region of chromosome 6, we hypothesized that a gene causing PCGwas present in this region, and that identification of the 6p25breakpoint from the balanced translocation patient would allow for theidentification of the gene responsible for PCG.

Mapping of the balanced translocation breakpoints. To facilitate theidentification and cloning of the t(6; 13) breakpoints, somatic cellhybrids were constructed from cell lines derived from the balancedtranslocation patient. Such hybrids are a useful tool in the mapping ofchromosomal rearrangements as they allow for the molecular analysis ofthe derivative chromosomes apart from their normal homologues. Twosomatic cell hybrids (H14 and H17) that each contained a singlederivative chromosome were identified by genotyping with highlypolymorphic markers. H17 was found to contain the derivative 13chromosome and the normal human chromosome 6. H14 was found to containthe derivative 6 chromosome in the absence of the normal 6 and 13chromosomes.

To map the t(6; 13) breakpoints, DNA from hybrids H14 and H17 along withDNA from controls (CEPH individuals 1331-01 and 1331-02, the balancedtranslocation patient and the hamster cell line, RJK88) were used as PCRtemplates to screen genetic markers to identify those markers flankingthe chromosome 6 and 13 breakpoints. Markers within the genetic map of6p25 were selected for the screen. The 6p25 breakpoint was found to belocated in a 5 cM region flanked by the markers D6S344 and D6S477.Similarly markers within the genetic map of 13q22 (Murray J. C. et al.,(1994) Science 265: 2049-2054) were evaluated. The chromosome 13q22breakpoint was found to be contained in 3 cM region flanked by themarkers D13S160 and D13S 170.

A high resolution physical map of the 6p25 region was constructed to aidin the cloning of the 6p breakpoint. This map, along with thedevelopment of STSs from YACs, allowed mapping of the breakpoint to asmall region near D6S344. BACs were then isolated from the regionsurrounding D6S344. Two BACs (185d15 and 471g19) were selected forsubcloning based on their ability to cover the region as determined bySTS content analysis. Primers derived from these BAC subclones werescreened by PCR using hybrid H14 as template. This allowedidentification of a clone that contained the 6p25 breakpoint. STScontent mapping within the clone as compared to hybrid H14 allowedprecise localization of the 6p25 breakpoint and obtainment of thesurrounding sequence. The junction fragment from the H14 hybrid DNA wasisolated using a primer flanking the 6p25 breakpoint in combination witha set of Alu-based primers (Dorin, J. R. et al., Hum. Mol. Genet. 1:53-59 (1992)). Sequence analysis of this fragment confirmed that it wasthe junction fragment from hybrid 1114. Since this junction fragmentcontained chromosome 13 sequence adjacent to the breakpoint, an STS wasdeveloped from this sequence and mapped onto the YAC/BAC contig of13q22. This STS mapped distal to the 13q22 breakpoint and its locationwithin the physical map of 13q22 was consistent with it being in closeproximity to the breakpoint. This marker also mapped to the BAC 163n9which had been isolated with markers that were proximal to thebreakpoint. This result indicated that BAC 163n9 contained the 13q22breakpoint of the balanced translocation patient.

Subclones from the 163n9 BAC were screened using the STS developed fromthe hybrid H14 junction fragment. A 3.5 kb subclone was identified thatcontained the 13q22 breakpoint. Sequence comparison with the normalchromosome 6 sequence and that from the hybrid H14 junction fragmentrevealed the location of the 13q22 breakpoint within the normal 13q22sequence. Finally, the junction fragment from the hybrid H17 wasevaluated to determine if there had been any gain or loss of material atthe site of the translocation. Using hybrid H17 DNA, a single fragmentwas generated by PCR using a primer proximal to the 13q22 breakpoint andone distal to the 6p25 breakpoint. Sequence analysis confirmed that thisfragment was the junction fragment from hybrid H17. Comparison of normalchromosome 6p25 sequence and normal chromosome 13q22 sequence alone withthat from the two junction fragments revealed that there had been a lossof 11 bp,

Identification of candidate genes within 6p25. Sequence generated fromboth sides of the 6p25 breakpoint (total of 10 kb) was analyzed for thepresence of gene sequences by using both BLASTN (Alstchul, S. F. et al.J. Mol. Biol. 215:403-410 (1990) and BLASTX (Gish, W et al Nat. Genet.3: 266-272 (1993)) to search public databases for homology to knowngenes. This sequence analysis resulted in the identification of a novelhuman gene showing strong homology to the GDP-Mannose 4,6-Dehydratasegene (E.C.4.2.1.47) that has been identified in a number of otherorganisms (Currie, H. L. et al., Clin. Diagn. Lab. Immunol. 2: 554-562(1995); Li. Y. et al., Virology 212: 134-150 (1995); Stevenson et al.,Bacteriol 178: 4885-4893 (1996); Bonin, C. P. et al. Proc. Natl. Acad.Sci. USA 94: 2085-2090 (1997)). By comparing the genomic sequence to thehuman cDNA sequence, the 6p25 breakpoint was localized to an intronupstream of the penultimate exon of this gene. Sequence analysis of BACscontaining this gene has been used to determine the partial intron/exonboundaries for this gene. Human mannose dehydratase appears to be 1.1 kbin size and has at least 7 exons. The genomic structure of two areas ofcoding sequence (345 and 253 bp) remain to be determined.

Physical mapping of the 6p25 region indicated that a human forkheadtranscription factor gene, FKHL7, is within 25 kb of the 6p25 breakpointand is translocated to the derivative 13 chromosome. Sequence of theforkhead domain of FKHL7 has been published (Pierrou, S. et al., EMBO J.13: 5002-5012 (1994)), along with FISH and somatic cell mapping datathat confirm the localization of this gene to 6p25.

To determine if a gene on chromosome 13 could be considered a candidategene for the glaucoma in the balanced translocation patient, 2 kb of DNAsurrounding the 13q22 breakpoint was sequenced. GRAIL (Xu, Y. et al.,Gen. Engin. 16: (241-253 (1994), and Uberbacher, E. C. and R. J. MuralProc. Natl. Acad. Sci. USA 88: 11261-11265 (1991))analysis of thissequence failed to find evidence for the presence of any predicted exonsin close proximity to the breakpoint. BLAST (Alstchul, S. F. et al., J.Mol. Biol. 215: 403-410 (1990). Gish, W. et al., Nat. Genet. 3: 266-272(1993)) analysis also failed to identify, any homologies to known genesor ESTs. The failure to detect a gene within the 13q22 region sequenceddoes not rule out the presence of a transcript as the possibility existsthat the 13q22 breakpoint has occurred within a large intron.

Analysis of the t(2:6) Unbalanced translocation, patient The patientwith the t(2,6′) unbalanced translocation is monosomic for a portion ofdistal 6p. In order to determine if this patient is monosomic for thet(6;13) breakpoint region of the balanced translocation patient, highlypolymorphic genetic markers were amplified using genomic DNA, from theunbalanced translocation patient as template. Markers proximal toD6S2652 were found to be heterozygous and markers distal to D6S2652 werefound to be homozygous. This indicates that mannose dehydratase andFKHL7 which are distal to D6S2652 are present in only a single copy inthe t(2.6) patient. Co-amplification of STSs in this region usingquantitative PCR confirms the loss of chromosomal material containingmannose dehydratase and FKHL7 in this patient.

Expression studies of FKHL7 and mannose dehydratase. In order toprioritize FKHL7 and mannose dehydratase as candidates for congenitalglaucoma, the expression pattern of each gene was evaluated by Northernblot analysis. Previous expression studies of FKHL7 demonstrated that a3.9 kb transcript was widely expressed in a variety of human adult andfetal tissues, while the expression of a second 3.4 kb transcript waslimited to fetal kidney (Pierrou, S et al., EMBO J. 13: 5002-5012(1994)). Northern blot analysis of a variety of human adult tissues(brain, heart, kidney, spleen, liver and colon) confirmed the broadexpression pattern of the 3.9 kb transcript and showed the co-expressionof a 3.0 kb transcript. These multiple FKHL7 transcripts may arise bydifferential polyadenylation, consistent with the presence of severalpolyadenylation signals in the FKHL7 3′UTR. Using a murine orthologue ofthe FKHL7 3′UTR, expression was analyzed in staged mouse embryos and invarious adult tissues including the eye. A 3.7 and 3.0 kb doublet wasmost abundantly expressed during embryogenesis, and of the adult tissuestested, expression in the eye and kidney were significantly higher thanthat seen in other adult tissues.

The expression pattern of mouse mannose dehydratase was also analyzed onthe identical Northern blot used for the FKHL7 experiments. A basallevel of expression was found during embryogenesis as well as in mostadult tissues, including the eye. The transcript size of mouse mannosedehydratase appears to be approximately 1.9 kb in size which is inagreement with the size predicted from the human mannose dehydratasecoding sequence.

Based on expression, both FKHL7 and mannose dehydratase are viablecandidate genes for causing glaucoma phenotypes. However, based on thehigher level of expression in the eye, the developmental regulation andputative function (Semina, E. V. et al., Nat. Genet. 14:392-399 (1966),Alward, W. L. M. et al., Am. J. Ophihalmol. 125:98-100 (1998), Glaser,T. et al., Nat. Genet. 2. 232-239 (1992); Jordan, T. et al., Nat. Genet.1: 328-332 11992) FKHL7 was favored as the better candidate.

Characterization of FKHL7 gene, FKHL7 is a monomeric DNA binding proteinthat shares a core binding site (RTAAAYA) with four other FKHL-likeproteins (Pierrou, S. et al., EMBO J. 13. 5002-5012 (1994). The forkheaddomain shows strong homology to the human gene, FKHL14, and the mousegenes Fkhl1 and FKHL14 by BLASTN (Altschul, S. F. et al., J. Mol. Biol.215: 403-410 (1990) analysis. A 9.8 kb subclone of 3AC 471g19 waspartially sequenced and determined to contain the entire coding regionof FKHL7 as well as 5′ and 3′ untranslated sequences. The human FKHL7coding sequence is 1.7 kb in size (553 amino acids) and contains nointrons. The 1659 bp open reading frame was found to contain thepreviously published DNA binding forkhead domain of FKHL7 (Pierrou, S.et al., EMBO J. 13: 5002-5012 (1994). The first in-frame ATG was foundto match well to the Kozak consensus sequence (Kozak, M. Annu. Rev.Cell. Biol. 8: 197-225 (1992); Kozak, M. Annu. Rev. Cell Biol. 8:197-225 (1992)). The COOH-terminal domain contains several stretches ofhomopolymeric runs of alanine and glycine. The FKHL7 coding regioncontains 5 recognition sites for the restriction enzyme NotI. The largenumber of NotI sites within the coding region of FKHL7 has adverselyaffected the identification of a fill-length cDNA since many cDNAlibraries are constructed with this restriction enzyme to preventcloning at an internal site. A BLASTN (Altschul, S. F. et al., J. Mol.Biol. 215: 403-410 (1990) screen of the public dbEST database with theFKHL7genomic sequence yields only partial human and mouse cDNA coverageof this gene. Based on the analysis of cDNA clones identified in thepublic databases, there is evidence for the utilization of at least twodifferent polyadenylation signals within the 3′ untranslated region.

Mutation screen. Although molecular analysis of the two translocationpatients was extremely useful for identifying FKHL7 and mannosedehydratase as candidates for causing glaucoma, neither gene wasconclusively demonstrated to be disease causing. Therefore, these twogenes were screened for mutations in a cohort of unrelated probands witheither PCG or anterior segment defects (RA and/or IH). Twenty-nineCaucasian probands were initially identified. Of these, 10 proved tohave SSCP evidence of a mutation in another glaucoma related gene(either CYP1B1 or P1TX2), and were therefore eliminated from the screen.The remaining 19 probands (6 PCG and 13 anterior chamber defectpatients) were screened by SSCP for mutations in mannose dehydratase andFKHL7. No mannose dehydratase mutations were identified in a screen of60% of the coding sequence of this gene. FKHL7 mutations were found infour probands and subsequently in related affected family members.

An 11 bp deletion upstream of the FKHL7 forkhead domain was identifiedin two brothers diagnosed with different anterior segment defects (RAand IH). Both brothers had glaucoma, and neither had the extra-ocularmanifestations of Reiger syndrome (RS). Their father was found to haveisolated posterior embryotoxon (PE), suggesting that the disease wasinherited through him as an autosomal dominant. He was also found tocarry the deletion. A second mutation was found in a proband and hermother who were both diagnosed with classic RA and glaucoma. Thismutation, a C to T transition within the forkhead domain causes a changefrom a serine to a leucine (SER131Leu). A third mutation, a C to Gtransversion within the forkhead domain, was found in a proband withsevere Axenfeld anomaly and glaucoma. This change results in thereplacement of isoleucine with methionine (Ile126MET) and is also foundin the father who was diagnosed with AA. Finally, a T to C transitionwas found in a proband of an extended family with a spectrum of anteriorsegment defects. This change results in the replacement of phenylalaninewith serine (Phe112Ser) within the forkhead domain. Three of themutations were not found in 128 unrelated normal individuals from anethnically similar control population (Caucasian). The fourth mutation(Phe112Ser) was only detected by direct sequencing of PCR products frompatient genomic DNA. This mutation was found to segregate with thedisease in an extended pedigree and was not present in an additional 12Caucasian individuals by sequence analysis.

The 11 bp deletion upstream of the FKHL7 forkhead domain is predicted tocause a truncated transcript (missing 477 aa) lacking the DNA-bindingforkhead domain. All three missense mutations occur within highlyconserved regions of the forkhead domain that has been implicated in theDNA binding properties of the molecule (Pierrou, S. et al, EMBOJ.13.5002-5012). The alteration of amino acids at these sites would beexpected to have an effect on the DNA binding specificity of FKHL7.Finally, screening of FKHL7 in the translocation patients failed toidentify mutations, suggesting that the presence of one abnormallyexpressed copy of the gene results in a disease phenotype.

20 1 2284 DNA Homo sapiens 1 cgagaaaagg tgacgcgggg cccgggcagg cggccggcgcgcggcccccc ccccccccgc 60 cctggttatt tggccgcctt cgccggcagc tcagggcagagtctcctgga aggcgcaggc 120 agtgtggcga gaagggcgcc tgcttgttct ttctttttgtctgctttccc ccgtttgcgc 180 ctggaagctg cgccgcgagt tcctgcaagg cggtctgccgcggccgggcc cggccttctc 240 ccctcgcagc gaccccgcct cgcggccgcg cgggccccgaggtagcccga ggcgccggag 300 gagccagccc cagcgagcgc cgggagaggc ggcagcgcagccggacgcac agcgcagcgg 360 gccggcacca gctcggccgg gcccggactc ggactcggcggccggcgcgg cgcggcccgg 420 cccgagcgag ggtggggggc ggcgggcggc gcggggcggcggcgagcggg ggcccacacc 480 ctcaaagccg aactaaatcg aaccccaaag caggaaaagctaaaggaacc catcaaggca 540 aaatcgaaac taaaaaaaaa aaatccaatt aaaaaaaacccctgagaata ttcaccacac 600 cagcgaacag aatatccctc caaaaattca gctcaccagcaccagcacga agaaaactct 660 attttcttaa ccgattaatt cagagccacc tccactttgccttgtctaaa taaacaaacc 720 cgtaaactgt tttatacaga gacagcaaaa tcttggtttattaaaggaca gtgttactcc 780 agataacacg taagtttctt cttgcttttc agagacctgctttcccctcc tcccgtctcc 840 cctctcttgc cttcttcctt gcctctcacc tgtaagatattattttatcc tatgttgaag 900 ggagggggaa agtccccgtt tatgaaagtc gctttctttttattcatgga cttgttttaa 960 aatgtaaatt gcaacatagt aatttatttt taatttgtagttggatgtcg tggaccaaac 1020 gccagaaagt gttcccaaaa cctgacgtta aattgcctgaaactttaaat tgtgcttttt 1080 ttctcattat aaaaagggaa actgtattaa tcttattctatcctcttttc tttctttttg 1140 ttgaacatat tcattgtttg tttattaata aattaccattcagtttgaat gagacctata 1200 tgtctggata ctttaataga gctttaatta ttacgaaaaaagatttcaga gataaaacac 1260 tagaagttac ctattctcca cctaaatctc tgaaaaatggagaaaccctc tgactagtcc 1320 atgtcaaatt ttactaaaag tctttttgtt tagatttattttcctgcagc atcttctgca 1380 aaatgtacta tatagtcagc ttgctttgag gctagtaaaaagatattttt ctaaacagat 1440 tggagttggc atataaacaa atacgttttc tcactaatgacagtccatga ttcggaaatt 1500 ttaagcccat gaatcagccg cggtcttacc acggtgatgcctgtgtgccg agagatggga 1560 ctgtgcggcc agatatgcac agataaatat ttggcttgtgtattccatat aaaattgcag 1620 tgcatattat acatccctgt gagccagatg ctgaatagattttttcctat tatttcagtc 1680 ctttataaaa ggaaaaataa accagttttt aaatgtatgtatataattct cccccattta 1740 caatccttca tgtattacat agaaggattg cttttttaaaaatatactgc gggttggaaa 1800 gggatattta atctttgaga aactatttta gaaaatatgtttgtagaaca attatttttg 1860 aaaaagattt aaagcaataa caagaaggaa ggcgagaggagcagaacatt ttggtctagg 1920 gtggtttctt tttaaaccat tttttcttgt taatttacagttaaacctag gggacaatcc 1980 ggattggccc tccccctttt gtaaataacc caggaaatgtaataaattca ttatcttagg 2040 gtgatctgcc ctgccaatca gactttgggg agatggcgatttgattacag acgttcgggg 2100 gggtgggggg cttgcagttt gttttggaga taatacagtttcctgctatc tgccgctcct 2160 atctagaggc aacacttaag cagtaattgc tgttgcttgttgtcaaaatt tgatcattgt 2220 taaaggattg ctgcaaataa atacacttta atttcagtcaaaaaaaaaaa aaaaaaaaaa 2280 aaaa 2284 2 553 PRT Homo sapiens 2 Met GlnAla Arg Tyr Ser Val Ser Ser Pro Asn Ser Leu Gly Val Val 1 5 10 15 ProTyr Leu Gly Gly Glu Gln Ser Tyr Tyr Arg Ala Ala Ala Ala Ala 20 25 30 AlaGly Gly Gly Tyr Thr Ala Met Pro Ala Pro Met Ser Val Tyr Ser 35 40 45 HisPro Ala His Ala Glu Gln Tyr Pro Gly Gly Met Ala Arg Ala Tyr 50 55 60 GlyPro Tyr Thr Pro Gln Pro Gln Pro Lys Asp Met Val Lys Pro Pro 65 70 75 80Tyr Ser Tyr Ile Ala Leu Ile Thr Met Ala Ile Gln Asn Ala Pro Asp 85 90 95Lys Lys Ile Thr Leu Asn Gly Ile Tyr Gln Phe Ile Met Asp Arg Phe 100 105110 Pro Phe Tyr Arg Asp Asn Lys Gln Gly Trp Gln Asn Ser Ile Arg His 115120 125 Asn Leu Ser Leu Asn Glu Cys Phe Val Lys Val Pro Arg Asp Asp Lys130 135 140 Lys Pro Gly Lys Gly Ser Tyr Trp Thr Leu Asp Pro Asp Ser TyrAsn 145 150 155 160 Met Phe Glu Asn Gly Ser Phe Leu Arg Arg Arg Arg ArgPhe Lys Lys 165 170 175 Lys Asp Ala Val Lys Asp Lys Glu Glu Lys Asp ArgLeu His Leu Lys 180 185 190 Glu Pro Pro Pro Pro Gly Arg Gln Pro Pro ProAla Pro Pro Glu Gln 195 200 205 Ala Asp Gly Asn Ala Pro Gly Pro Gln ProPro Pro Val Arg Ile Gln 210 215 220 Asp Ile Lys Thr Glu Asn Gly Thr CysPro Ser Pro Pro Gln Pro Leu 225 230 235 240 Ser Pro Ala Ala Ala Leu GlySer Gly Ser Ala Ala Ala Val Pro Lys 245 250 255 Ile Glu Ser Pro Asp SerSer Ser Ser Ser Leu Ser Ser Gly Ser Ser 260 265 270 Pro Pro Gly Ser LeuPro Ser Ala Arg Pro Leu Ser Leu Asp Gly Ala 275 280 285 Asp Ser Ala ProPro Pro Pro Ala Pro Ser Ala Pro Pro Pro His His 290 295 300 Ser Gln GlyPhe Ser Val Asp Asn Ile Met Thr Ser Leu Arg Gly Ser 305 310 315 320 ProGln Ser Ala Ala Ala Glu Leu Ser Ser Gly Leu Leu Ala Ser Ala 325 330 335Ala Ala Ser Ser Arg Ala Gly Ile Ala Pro Pro Leu Ala Leu Gly Ala 340 345350 Tyr Ser Pro Gly Gln Ser Ser Leu Tyr Ser Ser Pro Cys Ser Gln Thr 355360 365 Ser Ser Ala Gly Ser Ser Gly Gly Gly Gly Gly Gly Ala Gly Ala Ala370 375 380 Gly Gly Ala Gly Gly Ala Gly Thr Tyr His Cys Asn Leu Gln AlaMet 385 390 395 400 Ser Leu Tyr Ala Ala Gly Glu Arg Gly Gly His Leu GlnGly Ala Pro 405 410 415 Gly Gly Ala Gly Gly Ser Ala Val Asp Asn Pro LeuPro Asp Tyr Ser 420 425 430 Leu Pro Pro Val Thr Ser Ser Ser Ser Ser SerLeu Ser His Gly Gly 435 440 445 Gly Gly Gly Gly Gly Gly Gly Gly Gln GluAla Gly His His Pro Ala 450 455 460 Ala His Gln Gly Arg Leu Thr Ser TrpTyr Leu Asn Gln Ala Gly Gly 465 470 475 480 Asp Leu Gly His Leu Ala SerAla Ala Ala Ala Ala Ala Ala Ala Gly 485 490 495 Tyr Pro Gly Gln Gln GlnAsn Phe His Ser Val Arg Glu Met Phe Glu 500 505 510 Ser Gln Arg Ile GlyLeu Asn Asn Ser Pro Val Asn Gly Asn Ser Ser 515 520 525 Cys Gln Met AlaPhe Pro Ser Ser Gln Ser Leu Tyr Arg Thr Ser Gly 530 535 540 Ala Phe ValTyr Asp Cys Ser Lys Phe 545 550 3 1662 DNA Homo sapiens 3 atgcaggcgcgctactccgt gtccagcccc aactccctgg gagtggtgcc ctacctcggc 60 ggcgagcagagctactaccg cgcggcggcc gcggcggccg ggggcggcta caccgccatg 120 ccggcccccatgagcgtgta ctcgcaccct gcgcacgccg agcagtaccc gggcggcatg 180 gcccgcgcctacgggcccta cacgccgcag ccgcagccca aggacatggt gaagccgccc 240 tatagctacatcgcgctcat caccatggcc atccagaacg ccccggacaa gaagatcacc 300 ctgaacggcatctaccagtt catcatggac cgcttcccct tctaccggga caacaagcag 360 ggctggcagaacagcatccg ccacaacctc tcgctcaacg agtgcttcgt caaggtgccg 420 cgcgacgacaagaagccggg caagggcagc tactggacgc tggacccgga ctcctacaac 480 atgttcgagaacggcagctt cctgcggcgg cggcggcgct tcaagaagaa ggacgcggtg 540 aaggacaaggaggagaagga caggctgcac ctcaaggagc cgcccccgcc cggccgccag 600 cccccgcccgcgccgccgga gcaggccgac ggcaacgcgc ccggtccgca gccgccgccc 660 gtgcgcatccaggacatcaa gaccgagaac ggtacgtgcc cctcgccgcc ccagcccctg 720 tccccggccgccgccctggg cagcggcagc gccgccgcgg tgcccaagat cgagagcccc 780 gacagcagcagcagcagcct gtccagcggg agcagccccc cgggcagcct gccgtcggcg 840 cggccgctcagcctggacgg tgcggattcc gcgccgccgc cgcccgcgcc ctccgccccg 900 ccgccgcaccatagccaggg cttcagcgtg gacaacatca tgacgtcgct gcgggggtcg 960 ccgcagagcgcggccgcgga gctcagctcc ggccttctgg cctcggcggc cgcgtcctcg 1020 cgcgcggggatcgcaccccc gctggcgctc ggcgcctact cgcccggcca gagctccctc 1080 tacagctccccctgcagcca gacctccagc gcgggcagct cgggcggcgg cggcggcggc 1140 gcgggggccgcggggggcgc gggcggcgcc gggacctacc actgcaacct gcaagccatg 1200 agcctgtacgcggccggcga gcgcgggggc cacttgcagg gcgcgcccgg gggcgcgggc 1260 ggctcggccgtggacaaccc cctgcccgac tactctctgc ctccggtcac cagcagcagc 1320 tcgtcgtccctgagtcacgg cggcggcggc ggcggcggcg ggggaggcca ggaggccggc 1380 caccaccctgcggcccacca aggccgcctc acctcgtggt acctgaacca ggcgggcgga 1440 gacctgggccacttggcaag cgcggcggcg gcggcggcgg ccgcaggcta cccgggccag 1500 cagcagaacttccactcggt gcgggagatg ttcgagtcac agaggatcgg cttgaacaac 1560 tctccagtgaacgggaatag tagctgtcaa atggccttcc cttccagcca gtctctgtac 1620 cgcacgtccggagctttcgt ctacgactgt agcaagtttt ga 1662 4 106 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 4 Pro Lys Asp MetVal Lys Pro Pro Tyr Ser Tyr Ile Ala Leu Ile Thr 1 5 10 15 Met Ala IleGln Asn Ala Pro Asp Lys Lys Ile Thr Leu Asn Gly Ile 20 25 30 Tyr Gln PheIle Met Asp Arg Phe Pro Phe Tyr Arg Asp Asn Lys Gln 35 40 45 Gly Trp GlnAsn Ser Ile Arg His Asn Leu Ser Leu Asn Glu Cys Phe 50 55 60 Val Lys ValPro Arg Asp Asp Lys Lys Pro Gly Lys Gly Ser Tyr Trp 65 70 75 80 Thr LeuAsp Pro Asp Ser Tyr Asn Met Phe Glu Asn Gly Ser Phe Leu 85 90 95 Arg ArgArg Arg Arg Phe Lys Lys Lys Asp 100 105 5 106 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 5 Pro Lys Asp LeuVal Lys Pro Pro Tyr Ser Tyr Ile Ala Leu Ile Thr 1 5 10 15 Met Ala IleGln Asn Ala Pro Glu Lys Lys Ile Thr Leu Asn Gly Ile 20 25 30 Tyr Gln PheIle Met Asp Arg Phe Pro Phe Tyr Arg Glu Asn Lys Gln 35 40 45 Gly Trp GlnAsn Ser Ile Arg His Asn Leu Ser Leu Asn Glu Cys Phe 50 55 60 Val Lys ValPro Arg Asp Asp Lys Lys Pro Gly Lys Gly Ser Tyr Trp 65 70 75 80 Thr LeuAsp Pro Asp Ser Tyr Asn Met Phe Glu Asn Gly Ser Phe Leu 85 90 95 Arg ArgArg Arg Arg Phe Lys Lys Lys Asp 100 105 6 106 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 6 Thr Thr Glu ProThr Lys Pro Pro Tyr Ser Tyr Ile Ala Leu Ile Ala 1 5 10 15 Met Ala IleGln Ser Ser Pro Gly Gln Arg Ala Thr Leu Ser Gly Ile 20 25 30 Tyr Arg ValIle Met Gly Arg Phe Ala Phe Tyr Arg His Asn Arg Pro 35 40 45 Gly Trp GlnAsn Ser Ile Arg His Asn Leu Ser Leu Asn Glu Cys Phe 50 55 60 Val Lys ValPro Arg Asp Asp Arg Lys Pro Gly Lys Gly Ser Tyr Trp 65 70 75 80 Thr LeuAsp Pro Asp Cys His Asp Met Phe Glu His Gly Ser Phe Leu 85 90 95 Arg ArgArg Arg Arg Phe Thr Arg Gln Thr 100 105 7 106 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 7 Ala Glu Thr ProGln Lys Pro Pro Tyr Ser Tyr Ile Ala Leu Ile Ala 1 5 10 15 Met Ala IleGln Asp Ala Pro Glu Gln Arg Val Thr Leu Asn Gly Ile 20 25 30 Tyr Gln PheIle Met Asp Arg Phe Pro Phe Tyr His Asp Asn Arg Gln 35 40 45 Gly Trp GlnAsn Ser Ile Arg His Asn Leu Ser Leu Asn Asp Cys Phe 50 55 60 Val Lys ValPro Arg Glu Lys Gly Arg Pro Gly Lys Gly Ser Tyr Trp 65 70 75 80 Thr LeuAsp Pro Arg Cys Leu Asp Met Phe Glu Asn Gly Asn Tyr Arg 85 90 95 Arg ArgLys Arg Lys Pro Lys Pro Gly Pro 100 105 8 106 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 8 Pro Leu Gln ArgGly Lys Pro Pro Tyr Ser Tyr Ile Ala Leu Ile Ala 1 5 10 15 Met Ala LeuAla His Ala Pro Gly Arg Arg Leu Thr Leu Ala Ala Ile 20 25 30 Tyr Arg PheIle Thr Glu Arg Phe Ala Phe Tyr Arg Asp Ser Pro Arg 35 40 45 Lys Trp GlnAsn Ser Ile Arg His Asn Leu Thr Leu Asn Asp Cys Phe 50 55 60 Val Lys ValPro Arg Glu Pro Gly Asn Pro Gly Lys Gly Asn Tyr Trp 65 70 75 80 Thr LeuAsp Pro Ala Ala Ala Asp Met Phe Asp Asn Gly Ser Phe Leu 85 90 95 Pro ArgArg Lys Arg Phe Lys Arg Ala Glu 100 105 9 106 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 9 Pro Leu Gln ArgGly Lys Pro Pro Tyr Ser Tyr Ile Ala Leu Ile Ala 1 5 10 15 Met Ala IleAla His Ala Pro Glu Arg Arg Leu Thr Leu Gly Gly Ile 20 25 30 Tyr Lys PheIle Thr Glu Arg Phe Pro Phe Tyr Arg Asp Asn Pro Lys 35 40 45 Lys Trp GlnAsn Ser Ile Arg His Asn Leu Thr Leu Asn Asp Cys Phe 50 55 60 Leu Lys IlePro Arg Glu Ala Gly Arg Pro Gly Lys Gly Asn Tyr Trp 65 70 75 80 Ala LeuAsp Pro Asn Ala Glu Asp Met Phe Glu Ser Gly Ser Phe Leu 85 90 95 Arg ArgArg Lys Arg Phe Lys Arg Ser Asp 100 105 10 106 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 10 Ala Arg Gln ProAla Lys Pro Pro Ser Ser Tyr Ile Ala Leu Ile Thr 1 5 10 15 Met Ala IleLeu Gln Ser Pro His Lys Arg Leu Thr Leu Ser Gly Ile 20 25 30 Cys Ala PheIle Ser Asp Arg Phe Pro Tyr Tyr Arg Arg Lys Glu Pro 35 40 45 Gly Trp GlnAsn Ser Ile Arg His Asn Leu Ser Leu Asn Asp Cys Phe 50 55 60 Val Lys IlePro Arg Glu Pro Gly Arg Pro Gly Lys Gly Asn Tyr Trp 65 70 75 80 Ser LeuAsp Pro Ala Ser Gln Asp Met Phe Asp Asn Gly Ser Phe Leu 85 90 95 Arg ArgArg Lys Arg Phe Gln Arg Asn Gln 100 105 11 106 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 11 Arg Thr Arg LeuVal Lys Pro Pro Tyr Ser Tyr Ile Ala Leu Ile Thr 1 5 10 15 Met Ala IleLeu Gln Ser Pro Lys Lys Arg Leu Thr Leu Ser Glu Ile 20 25 30 Cys Glu PheIle Ser Gly Arg Phe Pro Tyr Tyr Arg Glu Lys Phe Pro 35 40 45 Ala Trp GlnAsn Ser Ile Arg His Asn Leu Ser Leu Asn Asp Cys Phe 50 55 60 Val Lys IlePro Arg Glu Pro Gly Asn Pro Gly Lys Gly Asn Tyr Trp 65 70 75 80 Thr LeuAsp Pro Glu Ser Ala Asp Met Phe Asp Asn Gly Ser Phe Leu 85 90 95 Arg ArgArg Lys Arg Phe Lys Arg Gln Pro 100 105 12 106 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 12 Arg Ser Pro LeuVal Lys Pro Pro Tyr Ser Tyr Ile Ala Leu Ile Thr 1 5 10 15 Met Ala IleLeu Gln Ser Pro Lys Lys Arg Leu Thr Leu Ser Glu Ile 20 25 30 Cys Glu PheIle Ser Gly Arg Phe Pro Tyr Tyr Arg Glu Lys Phe Pro 35 40 45 Ala Trp GlnAsn Ser Ile Arg His Asn Leu Ser Leu Asn Asp Cys Phe 50 55 60 Val Lys IlePro Arg Glu Pro Gly Asn Pro Gly Lys Gly Asn Tyr Trp 65 70 75 80 Thr LeuAsp Pro Glu Ser Ala Asp Met Phe Asp Asn Gly Ser Phe Leu 85 90 95 Arg ArgLys Arg Arg Phe Lys Arg Gln Pro 100 105 13 106 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 13 Ile Arg Arg ProGlu Lys Pro Pro Tyr Ser Tyr Ile Ala Leu Ile Val 1 5 10 15 Met Ala IleGln Ser Ser Pro Thr Lys Arg Leu Thr Leu Ser Glu Ile 20 25 30 Tyr Gln PheLeu Gln Ser Arg Phe Pro Phe Phe Arg Gly Ser Tyr Gln 35 40 45 Gly Trp LysAsn Ser Val Arg His Asn Leu Ser Leu Asn Glu Cys Phe 50 55 60 Ile Lys LeuPro Lys Gly Leu Gly Arg Pro Gly Lys Gly His Tyr Trp 65 70 75 80 Thr IleAsp Pro Ala Ser Glu Phe Met Phe Glu Asn Gly Ser Phe Arg 85 90 95 Arg ArgArg Arg Gly Phe Arg Arg Lys Cys 100 105 14 106 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 14 Leu Arg Arg ProGlu Lys Pro Pro Tyr Ser Tyr Ile Ala Leu Ile Val 1 5 10 15 Met Ala IleGln Ser Ser Pro Ser Lys Arg Leu Thr Leu Ser Glu Ile 20 25 30 Tyr Gln PheLeu Gln Ala Arg Phe Pro Phe Phe Arg Gly Ala Tyr Gln 35 40 45 Gly Trp LysAsn Ser Val Arg His Asn Leu Ser Leu Asn Glu Cys Phe 50 55 60 Ile Lys LeuPro Lys Gly Leu Gly Arg Pro Gly Lys Gly His Tyr Trp 65 70 75 80 Thr IleAsp Pro Ala Ser Glu Phe Met Phe Glu Asn Gly Ser Phe Arg 85 90 95 Arg ArgArg Arg Gly Phe Arg Arg Lys Cys 100 105 15 106 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 15 Asn Gly Lys TyrGlu Lys Pro Pro Phe Ser Tyr Asn Ala Leu Ile Met 1 5 10 15 Met Ala IleArg Gln Ser Pro Glu Lys Arg Leu Thr Leu Asn Gly Ile 20 25 30 Tyr Glu PheIle Met Lys Asn Phe Pro Tyr Tyr Arg Glu Asn Lys Gln 35 40 45 Gly Trp GlnAsn Ser Ile Arg His Asn Leu Ser Leu Asn Lys Cys Phe 50 55 60 Val Lys ValPro Arg His Tyr Asp Asp Pro Gly Lys Gly Asn Tyr Trp 65 70 75 80 Met LeuAsp Pro Ser Ser Tyr Asp Asp Val Ile Gly Gly Thr Thr Gly 85 90 95 Lys LeuArg Arg Arg Ser Thr Thr Ser Pro 100 105 16 106 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 16 Asn Gly Lys TyrGlu Lys Pro Pro Phe Ser Tyr Asn Ala Leu Ile Met 1 5 10 15 Met Ala MetArg Gln Ser Pro Glu Lys Arg Leu Thr Leu Asn Gly Ile 20 25 30 Tyr Glu PheIle Met Lys Asn Phe Pro Tyr Tyr Arg Glu Asn Lys Gln 35 40 45 Gly Trp GlnAsn Ser Ile Arg His Asn Leu Ser Leu Asn Lys Cys Phe 50 55 60 Val Lys ValPro Arg His Tyr Asp Asp Pro Gly Lys Gly Asn Tyr Trp 65 70 75 80 Met LeuAsp Pro Ser Ser Tyr Asp Asp Val Ile Gly Gly Thr Thr Gly 85 90 95 Lys LeuArg Arg Ser Thr Thr Ser Pro Ala 100 105 17 106 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 17 Gly Lys Tyr GluLys Pro Pro Pro Phe Ser Tyr Asn Ala Leu Ile Met 1 5 10 15 Met Ala IleArg Gln Ser Pro Glu Lys Arg Leu Thr Leu Asn Gly Ile 20 25 30 Tyr Glu PheIle Met Lys Asn Phe Pro Tyr Tyr Arg Glu Asn Lys Gln 35 40 45 Gly Trp HisAsn Ser Ile Arg Asp Asn Leu Ser Leu Asn Lys Cys Phe 50 55 60 Val Lys ValPro Arg His Tyr Asp Asp Pro Gly Lys Gly Asn Tyr Trp 65 70 75 80 Met LeuAsp Pro Ser Ser Asp Asp Val Phe Ile Gly Gly Thr Thr Gly 85 90 95 Lys LeuArg Arg Arg Ser Thr Thr Ser Arg 100 105 18 76 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 18 Leu Met Lys LeuVal Arg Pro Pro Tyr Ser Tyr Ser Ala Leu Ile Ala 1 5 10 15 Met Ala IleHis Gly Ala Pro Asp Lys Arg Leu Thr Leu Ser Gln Ile 20 25 30 Tyr Gln TyrVal Ala Asp Asn Phe Pro Phe Tyr Asn Lys Ser Lys Ala 35 40 45 Gly Trp GlnAsn Ser Ile Arg His Asn Leu Ser Leu Asn Asp Cys Phe 50 55 60 Lys Lys ValPro Arg Asp Glu Asp Asp Pro Gly Lys 65 70 75 19 106 PRT ArtificialSequence Description of Artificial Sequence Synthetic Peptide 19 Thr AsnPro His Val Lys Pro Pro Tyr Ser Tyr Ala Thr Leu Ile Cys 1 5 10 15 MetAla Met Gln Ala Ser Lys Ala Thr Lys Ile Thr Leu Ser Ala Ile 20 25 30 TyrLys Trp Ile Thr Asp Asn Phe Cys Tyr Phe Arg His Ala Asp Pro 35 40 45 ThrTrp Gln Asn Ser Ile Arg His Asn Leu Ser Leu Asn Lys Cys Phe 50 55 60 IleLys Val Pro Arg Glu Lys Asp Glu Pro Gly Lys Gly Gly Phe Trp 65 70 75 80Arg Ile Asp Pro Gln Tyr Ala Glu Arg Leu Leu Ser Gly Ala Phe Lys 85 90 95Lys Arg Arg Leu Pro Phe Val His Ile His 100 105 20 98 PRT ArtificialSequence Description of Artificial Sequence Synthetic Peptide 20 Trp GlyAsn Leu Ser Tyr Ala Asp Leu Ile Thr Lys Ala Ile Glu Ser 1 5 10 15 SerAla Glu Lys Arg Leu Thr Leu Ser Gln Ile Tyr Glu Trp Met Val 20 25 30 LysSer Val Pro Tyr Phe Lys Asp Lys Gly Asp Ser Asn Ser Ser Ala 35 40 45 GlyTrp Gln Lys Ser Ile Arg His Asn Leu Ser Leu His Ser Lys Phe 50 55 60 IleArg Val Gln Asn Glu Gly Thr Gly Lys Ser Ser Trp Trp Met Leu 65 70 75 80Asn Pro Glu Gly Gly Lys Ser Gly Lys Ser Pro Arg Arg Ala Ala Ser 85 90 95Met Asp

What is claimed is:
 1. A method for identifying a compound thatmodulates an FKHL7 DNA-binding activity, comprising the steps of: (a)contacting the compound with a cell or cellular extract, which expressesan FKHL7 gene product having the amino acid sequence of SEQ ID NO:2; and(b) determining the resulting FKHL7 DNA-binding activity, wherein anincrease or decrease in the FKHL7 DNA-binding activity in the presenceof the compound as compared to the DNA-binding activity in the absenceof the compound indicates that the compound is a modulator of FKHL7DNA-binding activity.
 2. The method of claim 1, wherein the compound isan agonist of an FKHL7 DNA-binding activity.
 3. The method of claim 1,wherein the compound is an antagonist of an FKHL7 DNA-binding activity.4. The method of claim 1, wherein the compound is selected from thegroup consisting of a polypeptide, a nucleic acid, a peptidomimetic, anda small molecule.
 5. The method of claim 4, wherein the small moleculeis a steroid.
 6. The method of claim 4, wherein the nucleic acid is amember selected from the group consisting of a gene replacement, anantisense, a ribozyme, and a triplex nucleic acid.
 7. A method foridentifying a compound that modulates FKHL7 DNA-binding activitycomprising the steps of: (a) combining an FKHL7 protein having the aminoacid sequence of SEQ ID NO:2, and FKHL7 binding partner, and a testcompound under conditions wherein, but for the test compound, the FKHL7protein and FKHL7 binding partner are able to interact; and (b)detecting the formation of an FKHL7 protein/FKH7 binding partnercomplex, such that a difference in the formation of an FKHL7protein/FKHL7 binding partner complex in the presence of a test compoundrelative to in the absence of the test compound indicates that the testcompound is a modulator of FKHL7 DNA-binding activity.
 8. The method ofclaim 7, wherein the test compound is selected from the group comprisinga polypeptide, a nucleic acid, a peptidomimetic, and a small molecule.9. The method of claim 8, wherein the small molecule is a steroid. 10.The method of claim 8, wherein the nucleic acid is a member selectedfrom the group consisting of a gene replacement, an antisense, aribozyme, and a triplex nucleic acid.
 11. The method of claim 7, whereinthe test compound is an agonist of on FKHL7 DNA-binding activity. 12.The method of claim 7, wherein the test compound is an antagonist of onFKHL7 DNA-binding activity.